IAP BIR domain binding compounds

ABSTRACT

Disclosed is an isomer, enantiomer, diastereoisomer or tautomer of a compound represented by Formula I or II 
                         
or a salt thereof, in which R 1 , R 2 , R 3 , R 100 , R 200 , R 300 , A, A 1 , BG, Q and Q 1  are substituents described herein. Also disclosed is the use of compounds of Formula I and II to treat proliferative disorders such as cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

Applicants hereby claim benefit from previously filed U.S. ProvisionalPatent Application Nos. 60/782,523, filed Mar. 16, 2006 and 60/876,994,filed Dec. 26, 2006, the entire contents of which are herebyincorporated by reference.

FIELD OF THE INVENTION

The present, invention concerns bridged compounds that bind to IAP BIRdomains, and which are useful for treating proliferative disorders anddisorders of dysregulated apoptosis, such as cancer.

BACKGROUND OF THE INVENTION

Apoptosis, or programmed cell death, typically occurs in the normaldevelopment and maintenance of healthy tissues in multicellularorganisms. It is a complex process which results in the removal ofdamaged, diseased or developmentally redundant cells, in the absence ofsigns of inflammation or necrosis.

Intrinsic apoptotic pathways are known to be dysregulated, mostparticularly in cancer and lymphoproliferative syndromes, as well asautoimmune disorders such as multiple sclerosis, in neurodegenerativediseases and in inflammation. As well, alterations in a host apoptoticresponse have been described in the development or maintenance of viraland bacterial infections.

The caspases are a family of proteolytic enzymes from the class ofcysteine proteases which are known to initiate and execute apoptosis. Innormal cells, the caspases are present as inactive zymogens, which arecatalytically activated following external signals, for example thoseresulting from ligand driven Death Receptor activation, such ascytokines or immunological agents, or by release of mitochondrialfactors, such as cytochrome C following genotoxic, chemotoxic, orradiation-induced cellular injury. The Inhibitors of Apoptosis Proteins(IAPs) constitute a family of proteins which are capable of binding toand inhibiting the caspases, thereby suppressing cellular apoptosis.Because of their central role in regulating Caspase activity, the IAPsare capable of inhibiting programmed cell death from a wide variety oftriggers, which include loss of homeostatic, or endogenous cellulargrowth control mechanisms, as well as chemotherapeutic drugs andirradiation.

The IAPs contain one to three homologous structural domains known asbaculovirus IAP repeat (BIR) domains. They may also contain a RING zincfinger domain at the C-terminus, with a capability of inducingubiquitinylation of IAP-binding molecules via its E3 ligase function.The human IAPs, XIAP, HIAP1 (also referred to as cIAP2), and HIAP2(cIAP1) each have three BIR domains, and a carboxy terminal RING zincfinger. Another IAP, NAIP, has three BIR domains (BIR1, BIR2 and BIR3),but no RING domain, whereas Livin, TsIAP and MLIAP have a single BIRdomain and a RING domain. The X chromosome-linked inhibitor of apoptosis(XIAP) is an example of an IAP which can inhibit the initiator caspase,known as caspase-9, and the effector caspases, Caspase-3 and Caspase-7,by direct binding. It can also induce the removal of caspases throughthe ubiquitylation-mediated proteasome pathway via the E3 ligaseactivity of a RING zinc finger domain. It is via the BIR3 domain thatXIAP binds to and inhibits caspase-9. The linker-BIR2 domain of XIAPinhibits the activity of caspases-3 and -7. The BIR domains have alsobeen associated with the interactions of IAPs with tumor necrosisfactor-receptor associated factor (TRAFs)-1 and -2, and to TAB1, asadaptor proteins effecting survival signaling through NFkB activation.The IAPs thus function as a direct brake on the apoptosis cascade, bypreventing the action of, or inhibiting active caspases and byre-directing cellular signaling to a pro-survival mode.

Progress in the cancer field has led to a new paradigm in cancer biologywherein neoplasia may be viewed as a failure of cancer cells to executenormal pathways of apoptosis. Normal cells receive continuous feedbackfrom their environment through various intracellular and extracellularfactors, and “commit suicide” if removed from this context. Thisinduction of apoptosis is achieved by activation of the caspase cascade.Cancer cells, however, gain the ability to overcome or bypass thisapoptosis regulation and continue with inappropriate proliferation. Themajority of treatments for cancer induce at least a partial apoptoticresponse in the cancer target cell, resulting in remission or initiationof tumor regression. In many cases, however, residual cells which areapoptosis-resistant are capable of escaping therapy and continuing theprocess of oncogenic/genetic change, resulting in the emergence ofhighly drug-resistant, metastatic disease which overcomes our ability toeffectively treat the disease. Furthermore, most cancer therapies,including radiation therapy and traditional chemotherapy do induceapoptosis in cancer cells, but cause additional cellular injury, due totheir lack of specificity in inducing apoptosis solely in cancer cells.The need to improve the specificity/potency of pro-apoptosis agents usedto treat cancer, and indeed other proliferative disorders, is importantbecause of the benefits in decreasing the side effects associated withadministration of these agents. Therefore, finding novel means ofinducing apoptosis in cancer cells is a highly desired medical need andits solution offers the possibility of entirely new treatments forcancer.

A growing body of data indicates that cancer cells may avoid apoptosisby the sustained over-expression of one or more members of the IAPfamily of proteins, as documented in many primary tumor biopsy samples,as well as most established cancer cell lines. Epidemiological studieshave demonstrated that over-expression of the various IAPs is associatedwith poor clinical prognosis and survival. For XIAP this is shown incancers as diverse as leukemia and ovarian cancer. Over expression ofHIAP1 and HIAP2 resulting from the frequent chromosome amplification ofthe 11q21-q23 region, which encompasses both, has been observed in avariety of malignancies, including medulloblastomas, renal cellcarcinomas, glioblastomas, and gastric carcinomas. (X)IAP negativeregulatory molecules such as XAF, appear to be tumor suppressors, whichare very frequently lost in clinical cancers. Thus, by their ability tosuppress the activation and execution of the intrinsic mediators ofapoptosis, the caspases, the IAPs may directly contribute to tumorprogression and resistance to pharmaceutical intervention. Induction ofapoptosis in cancer cells by the use of potent small molecules whichbind to specific IAP domains is the subject of this invention.

We and others have demonstrated the critical importance of theindividual BIR domains for affecting the antiapoptotic function of theIAPs. We have proposed that antagonists of the IAPs, which may bind tothe individual BIR domains, would disrupt the antiapoptotic function ofthe IAPs. Indeed, individual BIRs serve as critical binding sites forthe N-terminal Ser-Gly-Val-Asp, Ser-Gly-Pro-Ile and Ala-Thre-Pro-Ileresidues of the Caspases 3, 7, and 9, respectively, and such binding isimperative for the Caspase-inhibitory function of the IAPs. The bindingof N-terminal AxPy tetra-peptide residues to XIAP results in the releaseof the active caspases 3, 7 and 9. In the case of the other IAPs, suchas c-IAP1 and c-IAP2, the functions of the BIRs, when ligand-bound,appear to direct the activation of the ubiquitin ligase RING function ofthe IAPs to a bound target, or individual IAPs themselves, to causeproteosomal loss. In either case, small molecule antagonists of the IAPsshould be excellent pro-apoptotic agents, with potential uses in cancer,various proliferative disorders and inflammation.

A mammalian mitochondrial protein, namely Second Mitochondria-derivedActivator of Caspases (SMAC) which antagonizes IAP function, bindsmainly to the BIR 3 or 2 sites on respective IAPs via an AxPyamino-terminal tetrapeptide. Four Drosophila death-inducing proteins,Reaper, HID, Grim, and Sickle, which antagonize the ability of theDrosophila IAPs to inhibit caspases, also bind the BIR domains of theanalogous Drosophila IAPs via a short AxPy amino-terminal tetrapeptide,a sequence that fits into the BIR binding pocket and disruptsIAP-caspase interactions.

The overall topology of individual BIR domains is highly conservedbetween the human IAPs and between individual BIR domains of the humanIAPs, each BIR being a zinc finger polypeptide domain, locked into acoordinated Zn atom by two cysteines and a histidine residue. The X-raycrystallographic structures of XIAP BIR2 and BIR3 reveal a criticalbinding pocket for an AXPY motif on the surface of each BIR domain.There are alterations in the intervening amino acid sequences that formthe binding pocket and groove in both BIR2 and BIR3. Likewise, we havedescribed homologous domains in the BIRs of other IAPs cIAP1 and cIAP2.This opens the possibility of obtaining various classes of natural andsynthetic binding compounds which will have different specificity andbinding affinities between each of the BIR domains for each of the IAPs.Discerning the way in which such compounds will affect the biologicalfunction of the IAPs in cancer cells vs normal cells is a major newchallenge in the discovery of novel mechanism agents to treat cancer andother proliferative disorders where dysregulated IAP function isobserved. It is our finding that certain classes of BIR bindingcompounds may bind to IAP BIRs, with unexpected selectivity and potency,resulting in distinct therapeutic advantages for certain structuralclasses, potentially resulting from either IAP loss of function or lossof cellular IAP protein, or both.

A number of peptidic AxPy-like and heterocyclic modified AxPy peptidiccompounds have been described which activate cellular Caspase 3 byreportedly binding to XIAP BIR3. For a recent reviews, see Elmore etal., Annual Reports in Medicinal Chemistry, 40 (2006) 245-262; Sun etal., Bioorg. Med. Chem. Let. 15 (2005) 793-797; Oost et al., J. Med.Chem., 2004, 47(18), 4417-4426; Park et al., Bioorg. Med. Chem. Lett. 15(2005) 771-775; Franklin et al., Biochemistry, Vol. 42, No. 27, 2003,8223-8231; Kip et al., Biochemistry 2002, 41, 7344-7349; Wu et al.,Chemistry and Biology, Vol. 10, 759-767 (2003); Glover et al.,Analytical Biochemistry, 320 (2003) 157-169; United States publishedpatent application number 20020177557; and United States publishedpatent application number 20040180828; United States published patentapplication number US2006/0025347A1; United States published patentapplication number US2005/0197403A1; and United States published patentapplication number US2006/0194741A1.

The aforesaid compounds have been shown to target an isolated BIR3domain of XIAP via displacement of a fluorescently-labeled probe andthey appear to induce an apoptotic event in a select set of cancer celllines with potency in the low micromolar-nanomolar range. Thesecompounds displayed poor in-vivo activity, likely due to limitedbioavailability and may therefore have limited therapeutic application.

Thus, IAP BIR domains represent an attractive target for the discoveryand development of novel therapeutic agents, especially for thetreatment of proliferative disorders such as cancer.

SUMMARY OF THE INVENTION

The inventors have previously disclosed a series of compounds which bindto the BIR units of the IAPs and induce apoptosis in various cancer celllines (US published patent application number 20060264379). Acharacteristic of these compounds is the presence of a centralpyrrolidine unit. We now herein disclose that the linkage of two BIRbinding units via a substituted pyrrolidine, with preference for thesite, orientation and chemical nature of the linkage, provides novel anddistinctly advantageous classes of compounds with up to 1000 foldincrease in potency, resulting from induction of apoptosis, againstvarious cancer cell lines, over their corresponding non-bridged BIRbinding compounds and that these compounds display the requisitepotency, stability and pharmaceutical properties for the treatment ofhuman cancers. Advantageously, the chemical nature of the bridging groupcan be chosen to cause the translation of the high intrinsic(sub-nanomolar) cellular potency to microgram/kg potency in inhibitingand/or suppressing IAPs in several in-vivo xenograft models of humancancers. Furthermore, the compounds described have pharmaceuticallyacceptable stability in a range of mammalian tissues and fluids and havepharmaceutical properties which ensure adequate solubility andbioavailability using various routes of administration, suitable forclinical use. Such administration results in sustained in vivo effectsin mammals as measured in normal and tumor tissues.

In one embodiment of the present invention, there is provided an isomer,enantiomer, diastereoisomer or tautomer of a compound represented byFormula I or II:

wherein

-   m is 0, 1 or 2;-   Y is NH, O or S;-   BG is    -   1) —X-L-X¹—;-   X and X¹ are independently selected from    -   1) O,    -   2) NR¹³,    -   3) S,    -   4) —C₁-C₆ alkyl-,    -   5) —C₁-C₆ alkyl-O—,    -   6) —C₁-C₆ alkyl-NR¹³—,    -   7) —C₁-C₆ alkyl-S—,

-   L is selected from:    -   1) —C₁-C₂₀ alkyl-,    -   2) —C₂-C₆ alkenyl-,    -   3) —C₂-C₄ alkynyl-,    -   4) —C₃-C₇ cycloalkyl-,    -   5) -aryl-,    -   6) -biphenyl-,    -   7) -heteroaryl-,    -   8) -heterocyclyl-,    -   9) —C₁-C₆ alkyl-C₂-C₆ alkenyl)-C₁-C₆ alkyl-,    -   10) —C₁-C₆ alkyl-C₂-C₄ alkynyl)C₁-C₆ alkyl-    -   11) —C₁-C₆ alkyl-C₃-C₇ cycloalkyl)C₁-C₆ alkyl-,    -   12) —C₁-C₆ alkyl-aryl-C₁-C₆ alkyl-,    -   13) —C₁-C₆ alkyl-biphenyl-C₁-C₆ alkyl-,    -   14) —C₁-C₆ alkyl-heteroaryl-C₁-C₆ alkyl-,    -   15) —C₁-C₆ alkyl-heterocycyl-C₁-C₆ alkyl-,    -   16) —C₁-C₆ alkyl-Y—C₁-C₆ alkyl-,    -   17) -aryl-Y-aryl-,    -   18) -heteroaryl-Y-heteroaryl-,    -   19) -heterocyclyl-Y-heterocyclyl-,

wherein the alkyl, alkenyl, alkynyl and cycloalkyl are optionallysubstituted with one or more R⁶ substituents, and the aryl, biphenyl,heteroaryl, and heterocyclyl are optionally substituted with one or moreR¹⁰ substituents;

-   Q and Q¹ are independently selected from    -   1) NR⁴R⁵,    -   2) OR¹¹, or    -   3) S(O)_(m)R¹¹; or-   Q and Q¹ are independently selected from    -   1) aryl, or    -   2) heteroaryl, the aryl and the heteroaryl being optionally        substituted with one or more R¹⁰ substituents;-   A and A¹ are independently selected from    -   1) —CH₂—,    -   2) —CH₂CH₂—,    -   3) —CH(C₁-C₆ alkyl)-,    -   4) —CH(C₃-C₇ cycloalkyl-,    -   5) —C₃-C₇ cycloalkyl-,    -   6) —CH(C₁-C₆ alkyl-C₃-C₇ cycloalkyl)-,    -   7) —C(O)—, or    -   8) —C(O)OR¹³;-   R¹ and R¹⁰⁰ are independently selected from    -   1) H, or    -   2) C₁-C₆ alkyl optionally substituted with one or more R⁶        substituents;-   R² and R²⁰⁰ are independently selected from    -   1)H, or    -   2) C₁-C₆ alkyl optionally substituted with one or more R⁶        substituents;-   R³ and R³⁰⁰ are independently C₁-C₆ alkyl optionally substituted    with one or more R⁶ substituents;-   R⁴ and R⁵ are each independently selected from    -   1) H,    -   2) haloalkyl,    -   3) C₁-C₆ alkyl,    -   4) C₂-C₆ alkenyl,    -   5) C₂-C₄ alkynyl,    -   6) C₃-C₇ cycloalkyl,    -   7) C₃-C₇ cycloalkenyl,    -   8) aryl,    -   9) heteroaryl,    -   10) heterocyclyl,    -   11) heterobicyclyl,    -   12) —C(O)—R¹¹,    -   13) —C(O)O—R¹¹,    -   14) —C(═Y)NR⁸R⁹, or    -   15) —S(O)₂—R¹¹,        wherein the alkyl, alkenyl, alkynyl, cycloalkyl, and        cycloalkenyl are optionally substituted with one or more R⁶        substituents; and wherein the aryl, heteroaryl, heterocyclyl,        and heterobicyclyl are optionally substituted with one or more        R¹⁰ substituents;-   R⁶ is    -   1) halogen,    -   2) NO₂,    -   3) CN,    -   4) haloalkyl,    -   5) C₁-C₆ alkyl,    -   6) C₂-C₆ alkenyl,    -   7) C₂-C₄ alkynyl,    -   8) C₃-C₇ cycloalkyl,    -   9) C₃-C₇ cycloalkenyl,    -   10) aryl,    -   11) heteroaryl,    -   12) heterocyclyl,    -   13) heterobicyclyl,    -   14) OR⁷,    -   15) S(O)_(m)R⁷    -   16) NR⁸R⁹,    -   17) NR⁸S(O)₂R¹¹,    -   18) COR⁷,    -   19) C(O)OR⁷,    -   20) CONR⁸R⁹,    -   21) S(O)₂NR⁸R⁹    -   22) OC(O)R⁷,    -   23) OC(O)Y—R¹¹,    -   24) SC(O)R⁷, or    -   25) NC(Y)NR⁸R⁹,        wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl        is optionally substituted with one or more R¹⁰ substituents;-   R⁷ is    -   1) H,    -   2) haloalkyl,    -   3) C₁-C₆ alkyl,    -   4) C₂-C₆ alkenyl,    -   5) C₂-C₄ alkynyl,    -   6) C₃-C₇ cycloalkyl,    -   7) C₃-C₇ cycloalkenyl,    -   8) aryl,    -   9) heteroaryl,    -   10) heterocyclyl,    -   11) heterobicyclyl,    -   12) R⁸R⁹NC(═Y), or    -   13) C₁-C₆ alkyl-C₂-C₄ alkenyl, or    -   14) C₁-C₆ alkyl-C₂-C₄ alkynyl,        wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl is        optionally substituted with one or more R⁶ substituents; and        wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl        is optionally substituted with one or more R¹⁰ substituents;-   R⁸ and R⁹ are each independently    -   1) H,    -   2) haloalkyl,    -   3) C₁-C₆ alkyl,    -   4) C₂-C₆ alkenyl,    -   5) C₂-C₄ alkynyl,    -   6) C₃-C₇ cycloalkyl,    -   7) C₃-C₇ cycloalkenyl,    -   8) aryl,    -   9) heteroaryl,    -   10) heterocyclyl,    -   11) heterobicyclyl,    -   12) C(O)R¹¹,    -   13) C(O)Y—R¹¹, or    -   14) S(O)₂—R¹¹,        wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl is        optionally substituted with one or more R⁶ substituents; and        wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl        is optionally substituted with one or more R¹⁰ substituents;-   or R⁸ and R⁹ together with the nitrogen atom to which they are    bonded form a five, six or seven membered heterocyclic ring    optionally substituted with one or more R⁶ substituents;-   R¹⁰ is    -   1) halogen,    -   2) NO₂,    -   3) CN,    -   4) B(OR¹³)(OR¹⁴),    -   5) C₁-C₆ alkyl,    -   6) C₂-C₆ alkenyl,    -   7) C₂-C₄ alkynyl,    -   8) C₃-C₇ cycloalkyl,    -   9) C₃-C₇ cycloalkenyl,    -   10) haloalkyl,    -   11) OR⁷,    -   12) NR⁸R⁹,    -   13) SR⁷,    -   14) COR⁷,    -   15) C(O)O R⁷,    -   16) S(O)_(m)R⁷,    -   17) CONR⁸R⁹,    -   18) S(O)₂NR⁸R⁹,    -   19) aryl,    -   20) heteroaryl,    -   21) heterocyclyl, or    -   22) heterobicyclyl,        wherein the alkyl, alkenyl, alkynyl, cycloalkyl, and        cycloalkenyl is optionally substituted with one or more R⁶        substituents;-   R¹¹ is    -   1) haloalkyl,    -   2) C₁-C₆ alkyl,

3) C₂-C₆ alkenyl,

-   -   4) C₂-C₄ alkynyl,    -   5) C₃-C₇ cycloalkyl,    -   6) C₃-C₇ cycloalkenyl,    -   7) aryl,    -   8) heteroaryl,    -   9) heterocyclyl, or    -   10) heterobicyclyl,        wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl is        optionally substituted with one or more R⁶ substituents; and        wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl        is optionally substituted with one or more R¹⁰ substituents;

-   R¹² is    -   1) haloalkyl,    -   2) C₁-C₆ alkyl,    -   3) C₂-C₆ alkenyl,    -   4) C₂-C₄ alkynyl,    -   5) C₃-C₇ cycloalkyl,    -   6) C₃-C₇ cycloalkenyl,    -   7) aryl,    -   8) heteroaryl,    -   9) heterocyclyl,    -   10) heterobicyclyl,    -   11) C(O)—R¹¹,    -   12) C(O)O—R¹¹,    -   13) C(O)NR³R⁹,    -   14) S(O)_(m)—R¹¹, or    -   15) C(═Y)NR³R⁹,        wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl is        optionally substituted with one or more R⁶ substituents; and        wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl        is optionally substituted with one or more R¹⁰ substituents;

-   R¹³ and R¹⁴ are each independently    -   1) H, or    -   2) C₁-C₆ alkyl; or

-   R¹³ and R¹⁴ are combined to form a heterocyclic ring or a    heterobicyclyl ring;

-   R²⁰ is    -   1) H,    -   2) NH₂, or    -   3) NHFmoc;        or a prodrug; or the compound of Formula I or II is labeled with        a detectable label or an affinity tag.

In another aspect of the present invention, there is provided anintermediate compound represented by Formula 1-iv:

wherein PG³, R¹, R², R³, R⁴, and R⁵ are as defined herein.

In another aspect of the present invention, there is provided anintermediate compound represented by Formula 2-iv:

wherein PG⁴, R¹, R², R³, R⁴, and R⁵ are as defined herein.

In another aspect of the present invention, there is provided anintermediate compound represented by Formula 3-ii:

wherein PG⁴, R¹, R², R³, A, and Q are as defined herein, and L is—(CH₂)—, —(CH₂), —Y—(CH₂)_(r)—, -alkyl-aryl-alkyl-,-alkyl-heteroaryl-alkyl-, cycloalkyl, aryl or heteroaryl.

In another aspect of the present invention, there is provided anintermediate compound represented by Formula 4(i):

wherein PG⁴, PG⁴⁰⁰, L, R¹, R¹⁰⁰, R², R²⁰⁰, R³, R³⁰⁰, A, A¹, Q and Q¹ areas defined herein.

In another aspect of the present invention, there is provided anintermediate compound represented by Formula 6-ii:

wherein PG⁴, PG⁴⁰⁰, R¹, R¹⁰⁰, R², R²⁰⁰, R³, R³⁰⁰, A, A¹, Q and Q¹ are asdefined herein.

In another aspect of the present invention, there is provided anintermediate compound represented by Formula 7-v:

wherein PG⁴, PG⁴⁰⁰, L, R¹, R¹⁰⁰, R², R²⁰⁰, R³, R³⁰⁰, A, A¹, Q and Q¹ areas defined herein.

In another aspect of the present invention, there is provided anintermediate compound represented by Formula 8-ii:

wherein PG⁴ r, L, R¹⁰⁰, R²⁰⁰, A¹, and Q¹ are as defined herein.

In another aspect of the present invention, there is provided anintermediate compound represented by Formula 8-iii:

wherein PG⁴, r, L, R¹, R¹⁰⁰, R², R²⁰⁰, R³, A, A¹, Q and Q¹ are asdefined herein.

In another aspect of the present invention, there is provided anintermediate compound represented by Formula 17-i:

wherein PG⁴, L, R¹, R¹⁰⁰, R², R²⁰⁰, R³, R³⁰⁰, A, A¹, Q and Q¹ are asdefined herein.

In another aspect of the present invention, there is provided anintermediate compound represented by Formula 18-i:

wherein PG⁴, L, R¹, R¹⁰⁰, R², R²⁰⁰, R³, R³⁰⁰, A, A¹, Q and Q¹ are asdefined herein.

In another aspect of the present invention, there is provided anintermediate compound represented by Formula 19-2:

wherein PG¹, PG², L, X are as defined herein.

In another aspect of the present invention, there is provided anintermediate compound represented by Formula 19-3:

wherein PG², L, and X, are as defined herein.

In another aspect of the present invention, there is provided anintermediate compound represented by Formula 19-8:

wherein PG⁴, L, X, X¹, R¹, R¹⁰⁰, R², R²⁰⁰, R³, R³⁰⁰, R⁴, R⁴⁰⁰, R⁵ andR⁵⁰⁰ are as defined herein.

In another aspect of the present invention, there is provided anintermediate compound represented by Formula 20-1a:

wherein PG¹, L, X, X¹, R⁴ and R⁵ are as defined herein.

In another aspect of the present invention, there is provided anintermediate compound represented by Formula 20-2:

wherein PG⁴, L, X, and X¹, are as defined herein.

In another aspect of the present invention, there is provided anintermediate compound represented by Formula 20-4:

wherein PG⁴, L, X, X¹, R¹, R¹⁰⁰, R², R²⁰⁰, R³, R³⁰⁰, R⁴, R⁴⁰⁰, R⁵, andR⁵⁰⁰ are as defined herein.

In another aspect of the present invention, there is provided a processfor producing compounds represented by Formula I, described hereinabove,the process comprising:

-   a) coupling two intermediates represented by Formula 1-iv:

wherein PG³, R¹, R², R³, A, and Q are as defined herein,

-   in a solvent; and-   b) removing the protecting groups so as to form compounds of Formula    1.

In another aspect of the present invention, there is provided a processfor producing compounds represented by Formula I, described hereinabove,the process comprising:

-   a) coupling an intermediate represented by Formula 2-iv:

wherein PG⁴ is a protecting group, and R¹, R², R³, A, and Q are asdefined herein. and an activated diacid (0.5 equiv) in a solvent; and

b) removing the protecting groups so as to form compounds of Formula I.

In another aspect of the present invention, there is provided a methodfor the preparation of a pharmaceutically acceptable salt of compound offormula I and II, by the treatment of a compound of formula I or II with1 to 2 equiv of a pharmaceutically acceptable acid, as defined herein.

In another aspect of the present invention, there is provided apharmaceutical composition comprising a compound, as described above,mixed with a pharmaceutically acceptable carrier, diluent or excipient.

In another aspect of the present invention, there is provided apharmaceutical composition adapted for administration as an agent fortreating a proliferative disorder in a subject, comprising atherapeutically effective amount of a compound, as described above.

In another aspect of the present invention, there is provided apharmaceutical composition comprising a compound of Formula I incombination with one or more death receptor agonists, for example, anagonist of TRAIL receptor.

In another aspect of the present invention, there is provided apharmaceutical composition comprising a compound of formula I incombination with any therapeutic agent that increases the response ofone or more death receptor agonists, for example cytotoxic cytokinessuch as interferons.

In another aspect of the present invention, there is provided a methodof preparing a pharmaceutical composition, the method comprising: mixinga compound, as described above, with a pharmaceutically acceptablecarrier, diluent or excipient.

In another aspect of the present invention, there is provided a methodof treating a disease state characterized by insufficient apoptosis, themethod comprising: administering to a subject in need thereof, atherapeutically effective amount of a pharmaceutical composition, asdescribed above, so as to treat the disease state.

In another aspect of the present invention, there is provided a methodof modulating IAP function, the method comprising: contacting a cellwith a compound of the present invention so as to prevent binding of aBIR binding protein to an IAP BIR domain thereby modulating the IAPfunction.

In another aspect of the present invention, there is provided a methodof treating a proliferative disease, the method comprising:administering to a subject in need thereof, a therapeutically effectiveamount of the pharmaceutical composition, as described above, so as totreat the proliferative disease.

In another aspect of the present invention, there is provided a methodof treating cancer, the method comprising: administering to a subject inneed thereof, a therapeutically effective amount of the pharmaceuticalcomposition, as described above, so as to treat the cancer.

In another aspect of the present invention, there is provided a methodof treating cancer, the method comprising: administering to the subjectin need thereof, a therapeutically effective amount of a pharmaceuticalcomposition, as described above, in combination or sequentially with anagent selected from:

-   a) an estrogen receptor modulator,-   b) an androgen receptor modulator,-   c) retinoid receptor modulator,-   d) a cytotoxic agent,-   e) an antiproliferative agent,-   f) a prenyl-protein transferase inhibitor,-   g) an HMG-CoA reductase inhibitor,-   h) an HIV protease inhibitor,-   i) a reverse transcriptase inhibitor,-   k) an angiogenesis inhibitor,-   l) a PPAR-γ agonist,-   m) a PPAR-δ agonist,-   n) an inhibitor of inherent multidrug resistance,-   o) an anti-emetic agent,-   p) an agent useful in the treatment of anemia,-   q) agents useful in the treatment of neutropenia,-   r) an immunologic-enhancing drug,-   s) a proteasome inhibitor,-   t) an HDAC inhibitor,-   u) an inhibitor of the chymotrypsin-like activity in the proteasome,-   v) E3 ligase inhibitors,-   w) a modulator of the immune system such as, but not limited to,    interferon-alpha, Bacillus Calmette-Guerin (BCG), and ionizing    radition (UVB) that can induce the release of cytokines, such as the    interleukins, TNF, or induce release of death receptor ligands such    as TRAIL,-   x) a modulator of death receptors TRAIL and TRAIL receptor agonists    such as the humanized antibodies HGS-ETR1 and HGS-ETR2,    or in combination or sequentially with radiation therapy, so as to    treat the cancer.

In another aspect of the present invention, there is provided a methodfor the treatment or prevention of a proliferative disorder in asubject, the method comprising: administering to the subject atherapeutically effective amount of the composition, described above.

In another aspect of the present invention, the method further comprisesadministering to the subject a therapeutically effective amount of achemotherapeutic agent prior to, simultaneously with or afteradministration of the composition.

In yet another aspect, the method further comprises administering to thesubject a therapeutically effective amount of a death receptor agonistprior to, simultaneously with or after administration of thecomposition. The death receptor agonist is TRAIL or the death receptoragonist is a TRAIL antibody. The death receptor agonist is typicallyadministered in an amount that produces a synergistic effect.

In another aspect of the present invention, there is provided a probe,the probe being a compound of Formula I or II above, the compound beinglabeled with a detectable label or an affinity tag.

In another aspect of the present invention, there is provided a methodof identifying compounds that bind to an IAP BIR domain, the assaycomprising:

-   -   a) contacting an IAP BIR domain with a probe to form a probe:BIR        domain complex, the probe being displaceable by a test compound;    -   b) measuring a signal from the probe so as to establish a        reference level;    -   c) incubating the probe:BIR domain complex with the test        compound;    -   d) measuring the signal from the probe;    -   e) comparing the signal from step d) with the reference level, a        modulation of the signal being an indication that the test        compound binds to the BIR domain,        wherein the probe is a compound of Formula I or II labeled with        a detectable label or an affinity label.

In another aspect of the present invention, there is provided a methodof detecting loss of function or suppression of IAPs in vivo, the methodcomprising: a) administering to a subject, a therapeutically effectiveamount of a pharmaceutical composition, as defined above; b) isolating atissue sample from the subject; and c) detecting a loss of function orsuppression of IAPs from the sample.

BRIEF DESCRIPTION OF THE FIGURES

Further aspects and advantages of the present invention will becomebetter understood with reference to the description in association withthe following Figures, wherein:

FIG. 1 depicts SKOV-3 Human Ovarian Cancer Cell Line Xenograph Studywith compound 3. Female CD-1 nude mice (approximately 20-25 g) weresubcutaneously injected 5×10⁶ SKOV-3 human ovarian tumor cells in 50%matrigel subcutaneously in the right flank. On day 55, when tumors wereapproximately 100 mm³, treatment was initiated with compound 3 treatingwith compound for 5 consecutive days followed by 2 days with no drugtreatment for the duration of the experiment. Tumor size was measuredwith digital calipers and calculated as V=(a×b²)/2, wherein, a is thelongest dimension and b is the width. Tumor regression was observed at 1mg/kg while tumor stasis was observed to 0.3 mg/kg.

FIG. 2 depicts MDA-MB-231 Human Mammary Cancer Cell Line Xenograph Studywith compound 3. Female CD-1 nude mice (approximately 20-25 g) weresubcutaneously injected 1×10⁶ MDA-MB-231 human mammary tumor cells inthe right flank. On day 71, when tumors were approximately 90 mm³,treatment was initiated with compound 3 treating with compound for 5consecutive days followed by 2 days with no drug treatment for theduration of the experiment. Tumor size was measured with digitalcalipers and calculated as V=(a×b²)/2,

wherein, a is the longest dimension and b is the width. Tumor regressionwas observed at 1 mg/kg.

FIG. 3 demonstrates that compound 3 induces a loss of cIAP-1 in HCT-116cells in vitro. PC3, SKOV3, MDA-MB-231, HCT-116 cells were treated withvarious concentrations of compound 3 and incubated at 37° C. for 5hours. Cells were collected and the level of cIAP-1 and actin (loadingcontrol-not shown) were detected by western blot. Results indicated thatcompound 3 induced cIAP-1 loss in human cancer cells in a time dependentmanner (not shown). Using a similar method as described in FIG. 3,compound 3 induced a loss of loss of c-IAP1 from ES2 and 4T1 cell lines(not shown) and a loss of cIAP2 from PC3 cells (not shown).

FIG. 4 demonstrates the in vitro modulation of IAP's in mice whitecells. CD-1 mice whole blood was inclubated in vitro with variousconcentrations of compound 3 for 3 hours. White blood cells wereisolated from the treated whole blood via Ficoll gradient. Protein wasisolated from the white blood cells and the relative amount of cIAP-1and tubulin (loading control) was revealed by western blotting. In vitroresults indicated that compound 3 induces cIAP-1 loss in mice blood.

FIG. 5 demonstrates in vivo modulation of cIAP-1 in mice white cells.Compound 3 was administered to CD-1 mice by i.v. bolus injection as theindicated dose. After 1 to 48 hours the animals were sacrificed, theblood collected, the white cells were isolated on Ficoll gradient andthe protein extracted. The relative amount of cIAP-1 and tubulin(loading control) was revealed by western blotting (shown below at 3 hrtime point). Results indicated that compound 3 induces cIAP-1 downregulation in mice white cells, using ex-vivo detection methods.

FIG. 6 depicts a MDA-MB-231 Human Breast Cancer Cell Line XenographStudy with compound 24 (1 mg/kg). Female CD-1 nude mice (approximately20-25 g) were subcutaneously injected with 1×10⁶ MDA-MB-231 human breasttumor cells in 50% matrigel subcutaneously in the right flank. On day55, when tumors were approximately 100 mm³, treatment was initiated withcompound 24 treating with compound for 5 consecutive days followed by 2days with no drug treatment for the duration of the experiment. Tumorsize was measured with digital calipers and calculated as V=(a×b²)/2,wherein, a is the longest dimension and b is the width. Circles—20% HPCDcontrol; diamonds—compound 24.

FIG. 7 demonstrates in vivo modulation of cIAP-1 in rat white cells.Compound 24 was administered to rats by i.v. bolus injection. After 1 to48 hours, the blood was collected, the white cells were isolated onFicoll gradient and the protein extracted. The relative amount of cIAP-1and tubulin (loading control) was revealed by western blotting (shownbelow at 3 hr time point). Results indicated that compound 24 inducescIAP-1 loss in mice white cells, using ex-vivo detection methods.

DETAILED DESCRIPTION OF THE INVENTION

In many cancer and other diseases, an up-regulation of IAPs induced bygene defects or by chemotherapeutic agents has been correlated to anincreased resistance to apoptosis. Conversely, our results show thatcells decreased in IAP levels are more sensitive to chemotherapeuticagents and to death receptor agonists such as TRAIL. It is believed thata small molecule, which will antagonize IAP function, or a loss of IAPsfrom diseased cells, will be useful as a therapeutic agent. We reportherein that compounds of the instant invention can directly bind toIAPs, cause a down regulation of IAP proteins in cells, and induceapoptosis in cancer cells. Furthermore, compounds of the instantinvention have demonstrated synergistic effects in combination withclinically relavent agents used in the treatment of cancer.

We have discovered a novel series of bridged compounds, which bind tothe intact cellular IAPs and results in profound, sustained IAP proteindown-modulation and enhanced cellular apoptosis of cancer cells throughenhanced release of active Caspase 3. This biological response has beenobserved in various cell lines derived from human breast, pancreatic,colon, lung, ovarian cancers and primary human leukemia and lymphomacells. The compounds were found to be highly synergistic with DeathReceptor Agonist-mediated killing, such as TRAIL, TRAIL ReceptorMonoclonal Antibodies and TNF-α, in a large and comprehensive range ofcancer cells. Based upon these findings, the compounds will findapplication in treatment of many cancer types, such as solid tumors andtumors originating from the hematopoietic system. Moreover, thecompounds of the present invention may also find application inpreventing metastatic cancer cell invasion, inflammation, and in otherdiseases characterized by cells that are resistant to apoptosis viaupregulation of any one of the IAPs. As shown in FIG. 3, compound 3 iscapable of inducing the complete loss of c-IAP1/2 proteins from multipletumor cell lines at concentrations of less than 10 nM. Other compoundsof the instant invention were shown to display a similar ability toinduce time dependent IAP loss from cancer cells. This loss in IAPproteins strongly correlate to the ED₅₀ in SKOV3 cells.

The ‘bridging’ of two IAP BIR binding units, M1 and M2, described inmore detail below, using an appropriate ‘bridging unit’, linked to oneof the pyrrolidine rings, provides bridged IAP BIR binding compounds,which demonstrate significantly increased anti-cancer activity (10-1000fold), as compared to their monomeric units. This improved activityresults from an improved ability to bind to the BIR domains of theintact IAPs, and results in the induction of apoptosis in various cancercell lines.

Various factors influence the in vitro proapoptotic character of thecompounds of the present invention. Specifically, these include i) thepoint of attachment of the linker/pyrrolidine bond, ii) thestereochemistry at the linker/pyrrolidine bond, iii) the linker moietiesthemselves, including stereochemistry, regiochemistry, and the rigidityof the linker system, iv) alkyl substitution at R¹ and R¹⁰⁰, and v) thesubstitution pattern at R⁴, R⁴⁰⁰, R⁵, and R⁵⁰⁰.

For ease of description, throughout the description, the compounds ofFormula I and Formula 11, may also include the use of the terms P1, P2,P3, P4 and P5. These terms refer to the amino acids or modified aminoacids within either of Formula I or II. The following illustrates theuse of the terms:

wherein the waved line represents a covalent bond to another BIR bindingunit.

The compounds of the present invention may also be represented byFormula 3 or Formula 4 in which M1 and M2 represent independent BIRbinding domains.

wherein R¹, R², R¹⁰⁰, R²⁰⁰, R³, R³⁰⁰, R²⁰, A, A¹, Q, Q¹, and BG asdefined herein, and the dotted line represents a hypothetical dividingline for comparing the substituents associated with M1 and M2.

In one subset of Formula 3, M1 is the same as M2 and the dotted linedenotes a line of symmetry. In another subset, M1 is different from M2.

In one subset, compounds of Formula 4 are asymmetrical about the dottedline. In another subset the substituents on M1 and M2 are the same. Inanother subset, the substituents on M1 and M2 are different.

One skilled in the art will recognize that when M1 and M2 are the same,the R¹, R², R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹³, R¹⁴, r, m, Y, A, Q,and X substituents in M1 have the same meaning as the R¹⁰⁰, R²⁰⁰, R⁴,R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹³, R¹⁴, r, m, Y, A¹, Q¹, and X¹substituents repesctively in M2. When M1 and M2 are different, at leastone R¹, R², R¹⁰⁰, R²⁰⁰, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹³, R¹⁴, r,m, Y, A, A¹, Q, Q¹, X, and X¹ substituent is different in either of M1or M2.

Alternatively the substituents in M1 can be defined as R¹, R², R⁴, R⁵,R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹³, R¹⁴, r, m, p, Y, A, Q, and X, and thosein M2 can be defined as R¹⁰⁰, R²⁰⁰, R⁴⁰⁰, R⁵⁰⁰, R⁶⁰⁰, R⁷⁰⁰, R⁸⁰⁰, R⁹⁰⁰,R¹⁰⁰⁰, R¹¹⁰⁰, R¹³⁰⁰, R¹⁴⁰⁰, r¹, m¹, p¹, Y¹, A¹, Q¹ and X¹ respectively.In the case where M1 and M2 are the same, the R¹, R², R⁴, R⁵, R⁶, R⁷,R⁸, R⁹, R¹⁰, R¹¹, R¹³, R¹⁴, r, m, Y, A, Q, and X substituents in M1 havethe same meanings as R¹⁰⁰, R²⁰⁰, R⁴⁰⁰, R⁵⁰⁰, R⁶⁰⁰, R⁷⁰⁰, R⁸⁰⁰, R⁹⁰⁰,R¹⁰⁰⁰, R¹¹⁰⁰, R¹³⁰⁰, R¹⁴⁰⁰, r¹, m¹, Y¹, A¹, Q¹ and X¹ respectively inM2. In the case where M1 and M2 are different, at least one of theaforesaid substituents is different.

The compounds of the present invention are useful as BIR domain bindingcompounds in mammalian IAPs and are represented by either Formula I orFormula II. The following are embodiments, groups and substituents ofthe compounds according to Formula I and Formula II, which are describedhereinafter in detail.

-   A and A¹:

In one subset of compounds of Formula I or II, A and A¹ are both CH₂.

In an alternative subset of compounds of Formula I or II, A and A¹ areboth C═O.

In another alternative subset of compounds of Formula I or II, A is CH₂and A¹ is C═O.

In another alternative subset of compounds of Formula I or II, A and A¹are both C(O)OCH₃.

In another alternative subset of compounds of Formula I or II, A and A¹are both C(O)OH.

Any and each individual definition of A and A¹ as set out herein may becombined with any and each individual definition of Core, R¹, R², R¹⁰⁰,R²⁰⁰, R³, R³⁰⁰, Q, Q¹, and BG as set out herein.

Core:

Therefore, for compounds of Formula I, the present invention comprisescompounds of Formula 1A through 1C:

wherein BG, A, A¹, Q, Q¹, R¹, R¹⁰⁰, R², R²⁰⁰, R³, and R³⁰⁰ are asdefined hereinabove and hereinafter.

In one example, the present invention comprises compounds of Formula 1A.

In an alternative example, the present invention comprises compounds ofFormula 1 B.

In another alternative example, the present invention comprisescompounds of Formula 1C.

Alternatively, compounds of Formula II comprise compounds of Formula 2Aand 2B:

wherein BG, A, A¹, Q, Q¹, R¹, R¹⁰⁰, R², R²⁰⁰, R³, R³⁰⁰ and R²⁰ are asdefined hereinabove and hereinafter.

In one example, the present invention comprises compounds of Formula 2A.

Any and each individual definition of Core as set out herein may becombined with any and each individual definition of A, A¹, R¹, R², R¹⁰⁰,R²⁰⁰, R³, R³⁰⁰, R²⁰, Q. Q¹, and BG as set out herein.

BG:

In one subset of the aforesaid compounds, BG is —X-L-X¹—.

In one subset, for compounds of Formula I in which BG is —X-L-X¹—, theinvention comprises compounds of Formula 1a through 1c:

wherein L, X, X¹, A, A¹, Q, Q¹, R¹, R¹⁰⁰, R², R²⁰⁰, R³, and R³⁰⁰ are asdefined hereinabove and hereinafter.

One further subset of the aforesaid compounds comprises compounds ofFormula 1.1a through 1.1c:

wherein L, X, X¹, A, A¹, R¹, R¹⁰⁰, R², R²⁰⁰, R³, R³⁰⁰, R⁴, R⁴⁰⁰, R⁵ andR⁵⁰⁰ are as defined hereinabove and hereinafter.

In one subset, for compounds of Formula II in which BG is —X-L-X¹—, theinvention comprises compounds of Formula 2a:

wherein L, X, X¹, A, A¹, Q, Q¹, R¹, R¹⁰⁰, R², R²⁰⁰, R³ and R²⁰ are asdefined hereinabove and hereinafter.

Any and each individual definition of BG as set out herein may becombined with any and each individual definition of Core, R¹, R², R¹⁰⁰,R²⁰⁰, R³, R³⁰⁰, A, A¹, Q, and Q¹ as set out herein.

-   X and X¹:

In one subset of the aforesaid compounds, X and X¹ are independentlyselected from

-   -   1) O,    -   2) NR¹³,    -   3) S,    -   4) C₁-C₆ alkyl-O—,    -   5) C₁-C₆ alkyl,

In one example X and X¹ are independently selected from:

Any and each individual definition of X and X¹ as set out herein may becombined with any and each individual definition of Core, L, A, A¹, R¹,R², R¹⁰⁰, R²⁰⁰, R³, R³⁰⁰, R²⁰, Q, Q¹, and BG as set out herein.

-   L:

In one subset of the aforesaid compounds, L is selected from:

-   -   1) —C₁-C₂₀ alkyl-,    -   2) —C₃-C₇ cycloalkyl-,    -   3)-aryl-,    -   4)-biphenyl-,    -   5)-heteroaryl-,    -   6) —C₁-C₆ alkyl-(C₂-C₄ alkynyl)-C₁-C₆ alkyl-    -   7) —C₁-C₆ alkyl-aryl-C₁-C₆ alkyl-,    -   8)-aryl-Y-aryl-,

wherein the alkyl and cycloalkyl are optionally substituted with one ormore R⁶ substituents, and the aryl, biphenyl and heteroaryl areoptionally substituted with one or more R¹⁰ substituents.

Typical Examples of L Include

Any and each individual definition of L as set out herein may becombined with any and each individual definition of Core, A, A¹, r, R¹,R²R¹⁰⁰, R²⁰⁰, R³, R³⁰⁰, R²⁰, X, X¹, Q, or Q¹ as set out herein.

-   r:

In the aforesaid aspect, r is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 or10.

Any and each individual definition of r as set out herein may becombined with any and each individual definition of Core, A, L, A¹, R¹,R², R¹⁰⁰, R²⁰⁰, R³, R³⁰⁰, R²⁰, Q, Q¹, X and X¹ as set out herein.

More explicitly, the invention comprises compounds of Formula 1.1through 1.18:

wherein r, A, A¹, Q. Q¹, R¹, R¹⁰⁰, R², R²⁰⁰, R³ and R³⁰⁰ are as definedhereinabove.

Alternatively more explicitly, the invention comprises compounds ofFormula 2.1 and 2.2:

wherein A, A¹, Q, Q¹, R¹, R¹⁰⁰, R², R²⁰⁰, R³ and R²⁰ are as definedhereinabove.

-   R¹ and R¹⁰⁰:

In one subset of the aforesaid compounds R¹ and R¹⁰⁰ are both H.

In one subset of the aforesaid compounds R¹ and R¹⁰⁰ are both C₁-C₆alkyl. In one example, R¹ and R¹⁰⁰ are both CH₃.

Any and each individual definition of R¹ and R¹⁰⁰ as set out herein maybe combined with any and each individual definition of Core, A, A¹, R²,R²⁰⁰, R³, R³⁰⁰, Q, Q¹, B, B¹, and BG as set out herein.

-   R² and R²⁰⁰:

In one subset of the aforesaid compounds R² and R²⁰⁰ are both C₁-C₆alkyl optionally substituted with OH.

In one example, R² and R²⁰⁰ are both CH₃.

In another example, R² is CH₂OH and R³⁰⁰ is CH₃.

In another example, R² and R²⁰⁰ are both CH₂OH.

In another example, R² and R²⁰⁰ are both CH₂CH₃.

Any and each individual definition of R² and R²⁰⁰ as set out herein maybe combined with any and each individual definition of Core, A, A₁, R¹,R¹⁰⁰, R³, R³⁰⁰, Q, Q¹, and BG as set out herein.

-   R³ and R³⁰⁰:

In one subset of compounds of Formula I, R³ and R³⁰⁰ are both C₁-C₆alkyl.

In one example, R³ and R³⁰⁰ are both C(CH₃)₃.

In subset of compounds of Formula II, R³ is C₁-C₆ alkyl. In one example,R³ is C(CH₃)₃.

Any and each individual definition of R³ and R³⁰⁰ as set out herein maybe combined with any and each individual definition of Core, A, A¹, R¹,R¹⁰⁰, R², R²⁰⁰, Q, Q¹, and BG as set out herein.

-   Q and Q¹:

In one subset of the aforesaid compounds, Q and Q¹ are both NR⁴R⁵,wherein R⁴ and R⁵ are as defined herein.

Any and each individual definition of Q and Q¹ as set out herein may becombined with any and each individual definition of Core, A, A¹, R¹,R¹⁰⁰, R², R²⁰⁰, R³, R³⁰⁰ and BG as set out herein.

-   R⁴ and R⁵:

In one subset of the aforesaid compounds in which A and A¹ are both C═O,R⁴ is H and R⁵ is selected from

-   -   1) C₁-C₆ alkyl    -   2) C₂-C₆ alkenyl,    -   3) C₂-C₄ alkynyl,    -   4) C₃-C₇ cycloalkyl,    -   5) C₃-C₇ cycloalkenyl,    -   6) aryl,    -   7) heteroaryl,    -   8) heterocyclyl, or    -   9) heterobicyclyl,        wherein the alkyl, alkenyl, alkynyl, cycloalkyl, and        cycloalkenyl are optionally substituted with one or more R⁶        substituents; and wherein the aryl, heteroaryl, heterocyclyl,        and heterobicyclyl are optionally substituted with one or more        R¹⁰ substituents;        wherein R⁶ and R¹⁰ are as defined herein.

In another subset of the above compounds, R⁴ is H and R⁵ is selectedfrom:

-   -   1) C₁-C₆ alkyl, or    -   2) aryl,        wherein the alkyl is optionally substituted with one or two R⁶        substituents; and        wherein the aryl is optionally substituted with one R¹⁰        substituent;        wherein R⁶ and R¹⁰ are as defined herein.

Examples of the aforesaid subset include, R⁴ is H and R⁵ is selectedfrom the group consisting of:

Therefore, when A and A¹ are both C═O, then Q and Q¹ are both

In another example, Q and Q¹ are both

In another example, Q is

and Q¹ is

In another example, Q and Q¹ are both

In another example, Q and Q¹ are both

In another example, Q and Q¹ are both

In another example, Q and Q¹ are both

In another example, Q and Q¹ are both

In another example, Q and Q¹ are both

In another example, Q and Q¹ are both

In another example, Q and Q¹ are both

In another example, Q and Q¹ are both

In another example, Q and Q¹ are both

In another example, Q and Q¹ are both

In another example, Q and Q¹ are both

In another example, Q and Q¹ are both

In another example, Q and Q¹ are both

In another example, Q and Q¹ are both

In another example, Q and Q¹ are both

In another example, Q and Q¹ are both

In another example, Q and Q¹ are both

In another example, Q and Q¹ are both

In another example, Q and Q¹ are both

In another example, Q and Q¹ are both

In another example, Q and Q¹ are both

In an alternative subset of the aforesaid compounds in which A and A¹are both CH₂, then R⁴ and R⁵ are each independently

-   -   1) haloalkyl,    -   2) C₁-C₆ alkyl,    -   3) C₂-C₆ alkenyl,    -   4) C₂-C₄ alkynyl,    -   5) C₃-C₇ cycloalkyl,    -   6) C₃-C₇ cycloalkenyl,    -   7) aryl,    -   8) heteroaryl,    -   9) heterocyclyl,    -   10) heterobicyclyl,    -   11) —C(O)—R¹¹,    -   12) —C(O)O—R¹¹,    -   13) —C(═Y)NR⁸R⁹, or    -   14) —S(O)₂—R¹¹,        wherein the alkyl, alkenyl, alkynyl, cycloalkyl, and        cycloalkenyl are optionally substituted with one or more R⁶        substituents; and wherein the aryl, heteroaryl, heterocyclyl,        and heterobicyclyl are optionally substituted with one or more        R¹⁰ substituents;        wherein Y, R⁶, R⁸, R⁹, R¹⁰ and R¹¹ are as defined herein.

In another subset of the above compounds, R⁴ and R⁵ are independentlyselected from

-   -   1) C₁-C₆ alkyl,    -   2) —C(O)—R¹¹,    -   3) —C(O)O—R¹¹, or    -   4) —S(O)₂—R¹¹,        wherein the alkyl is substituted with an R⁶ substituent;        wherein R⁶ and R¹¹ are as defined herein.

In one subset of the aforesaid compounds, R⁴ is S(O)₂CH₃ and R⁵ is

In another subset of the aforesaid compounds, R⁴ is C(O)CH₃ and R⁵ is

In another subset of the aforesaid compounds, R⁴ is

and R⁵ is

Any and each individual definition of R⁴ and R⁵ as set out herein may becombined with any and each individual definition of Core, A, A¹, R¹,R¹⁰⁰, R², R²⁰⁰, R³, R³⁰⁰, and BG as set out herein.

-   R¹¹:

In one subset of the aforesaid compounds,

-   R¹¹ is    -   1) C₁-C₆ alkyl, or    -   2) aryl,        wherein the alkyl is optionally substituted with one or more R⁶        substituents; and wherein the aryl is optionally substituted        with one or more R¹⁰ substituents;        wherein R⁶ and R¹⁰ are as defined herein.

In one subset of the aforesaid compounds, R¹¹ is

-   -   1) C₁-C₆ alkyl optionally substituted with one or two R⁶        substituents, or    -   2) phenyl optionally substituted with one R¹⁰ substituent;        wherein the R⁶ and the R¹⁰ substituents are as defined herein.

Any and each individual definition of R¹¹ as set out herein may becombined with any and each individual definition of Core, A, A¹, R¹,R¹⁰⁰, R², R²⁰⁰, R⁴, R⁵, R³, R³⁰⁰ and BG as set out herein.

-   R⁶:

In one subset of the aforesaid compounds, R⁶ is

-   -   1) halogen,    -   2) NO₂,    -   3) CN,    -   4) aryl,    -   5) heteroaryl,    -   6) heterocyclyl,    -   7) heterobicyclyl,    -   8) OR⁷,    -   9) SR⁷, or    -   10) NR⁸R⁹,        wherein the aryl, heteroaryl, heterocyclyl, and heterobicyclyl        is optionally substituted with one or more R¹⁰ substituents;        wherein R⁷, R⁸, R⁹ and R¹⁰ are as defined herein.

In another subset of the aforesaid compounds, R⁶ is

-   -   1) halogen,    -   2) aryl, or    -   3) NR⁸R⁹,        wherein the aryl is optionally substituted with one R¹⁰        substituent;        wherein R⁸, R⁹ and R¹⁰ are as defined herein.

In one subset of the aforesaid compounds, R⁶ is

-   -   1) halogen,    -   2) phenyl, or    -   3) NR⁸R⁹,        wherein the phenyl is optionally substituted with one R¹⁰        substituent;        wherein R⁸ and R⁹ are as defined herein.

Any and each individual definition of R⁶ as set out herein may becombined with any and each individual definition of Core, A, A¹, R¹,R¹⁰⁰, R², R²⁰⁰, R⁴, R⁵, R³, R³⁰⁰ and BG as set out herein.

-   R⁸ and R⁹:

In one subset of the aforesaid compounds, R⁸ and R⁹ are eachindependently

-   -   1) H,    -   2) haloalkyl,    -   3) C₁-C₆ alkyl,    -   4) C₂-C₆ alkenyl,    -   5) C₂-C₄ alkynyl,    -   6) C₃-C₇ cycloalkyl, or    -   7) C₃-C₇ cycloalkenyl,        wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl is        optionally substituted with one or more R⁶ substituents;        wherein the R⁶ substituents are as defined herein.

In another subset of the aforesaid compounds, R⁸ and R⁹ are eachindependently

-   -   1) H, or    -   2) C₁-C₆ alkyl,        wherein the alkyl is optionally substituted with an aryl.

Any and each individual definition of R⁸ and R⁹ as set out herein may becombined with any and each individual definition of Core, A, A¹, R¹,R¹⁰⁰, R², R²⁰⁰, R⁴, R⁵, R³, R³⁰⁰ and BG as set out herein.

-   R¹⁰:

In one aspect of the aforesaid compounds, R¹⁰ is

-   -   1) halogen,    -   2) NO₂,    -   3) CN,    -   4) haloalkyl,    -   5) OR⁷,    -   6) NR⁸R⁹, or    -   7) SR⁷;        wherein R⁷, R⁸, and R⁹ are as defined herein.

In another aspect of the aforesaid compounds, R¹⁰ is

-   -   1) halogen, or    -   2) OC₁-C₆ alkyl.

Any and each individual definition of R¹⁰ as set out herein may becombined with any and each individual definition of Core, A, A¹, R¹,R¹⁰⁰, R², R²⁰⁰, R⁴, R⁵, R³, R³⁰⁰ and BG as set out herein.

Alternatively, the invention provides an isomer, enantiomer,diastereoisomer or tautomer of a compound represented by Formula I orFormula 2:

wherein

-   n is 0 or 1;-   m is 0, 1 or 2;-   p is 1 or 2;-   Y is NH, O or S;-   LG is    -   2) —X-L-X¹-;-   X and X¹ are independently selected from    -   1) O,    -   2) NR¹³,    -   3) S,    -   4) C₁-C₆ alkyl-O—,    -   5) C₁-C₆ alkyl-NR¹³—,    -   6) C₁-C₆ alkyl-S—,    -   7) C₁-C₆ alkyl-aryl-O—

-   L is selected from:    -   1) —C₁-C₂₀ alkyl-,    -   2) —C₂-C₆ alkenyl-,    -   3) —C₂-C₄ alkynyl-,    -   4) —C₃-C₇ cycloalkyl-,    -   5) -phenyl-,    -   6) -biphenyl-,    -   7) -heteroaryl-,    -   8) -heterocycyl-,    -   9) —C₁-C₆ alkyl-C₂-C₆ alkenyl)-C₁-C₆ alkyl-,    -   10) —C₁-C₆ alkyl-C₂-C₄ alkynyl)-C₁-C₆ alkyl,    -   11) —C₁-C₆ alkyl-C₃-C₇ cycloalkyl)-C₁-C₆ alkyl,    -   12) —C₁-C₆ alkyl-phenyl-C₁-C₆ alkyl,    -   13) —C₁-C₆ alkyl-biphenyl-C₁-C₆ alkyl,    -   14) —C₁-C₆ alkyl-heteroaryl-C₁-C₆ alkyl,    -   15) —C₁-C₆ alkyl heterocycyl-C₁-C₆ alkyl,    -   16) —C₁-C₆ alkyl-O—C₁-C₆ alkyl,    -   17) —C(O)-aryl-C(O)—,    -   18) —C(O)-heteroaryl-C(O)—,    -   19) —C(O)—(C₁-C₆ alkyl)-aryl-(C₁-C₆ alkyl)-C(O)—,    -   20) —C(O)— (C₁-C₆ alkyl)-heteroaryl-(C₁-C₆ alkyl)-C(O)—, or    -   21) —C(O)— (C₁-C₆ alkyl(C₃-C₇ cycloalkyl) (C₁-C₆ alkyl)-C(O)—;

Q and Q¹ are independently selected from

-   -   1) NR⁴R⁵,    -   2) OR¹¹, or    -   3) S(O)_(m)R¹¹; or

-   Q and Q¹ are independently selected from aryl or heteroaryl, being    optionally substituted with R¹² substituents; or

-   Q and Q¹ are independently

wherein G is a 5, 6 or 7 membered ring which optionally incorporates oneor more heteroatoms selected from S, N or O, and which is optionallysubstituted with one or more R¹² substituents, the ring being optionallyfused with an aryl or a heteroaryl, the aryl and the heteroaryl beingoptionally substituted with one or more R¹² substituents;

-   A and A¹ are independently selected from    -   1) —CH₂—,    -   2) —CH₂CH₂—,    -   3) —CH(C₁-C₆ alkyl),    -   4) —CH(C₃-C₇ cycloalkyl),    -   5) —C₃-C₇ cycloalkyl-,    -   6) —CH(C₁-C₆ alkyl-C₃-C₇ cycloalkyl), or    -   7) —C(O)—;-   R¹ and R¹⁰⁰ are independently selected from    -   3) H, or    -   4) C₁-C₆ alkyl optionally substituted with one or more R⁶        substituents;-   R² and R²⁰⁰ are independently H or C₁-C₆ alkyl optionally    substituted with one or more R⁶ substituents;-   R⁴ and R⁵ are independently selected from:    -   1) H,    -   2) C₁-C₆ alkyl,    -   3) C₃-C₇ cycloalkyl,    -   4) haloalkyl,    -   5) aryl,    -   6) biphenyl,    -   7) heteroaryl-aryl,    -   8) aryl-heteroaryl,    -   9) aryl-heterocyclyl,    -   10) heterocyclyl,    -   11) heteroaryl,    -   12) heterocyclyl,    -   13) C₁-C₆ alkyl-O_(n)C(O)—,    -   14) haloalkyl-O_(n)C(O)—,    -   15) C₃-C₇ cycloalkyl-O_(n)C(O)—,    -   16) aryl-O_(n)C(O)—,    -   17) heteroaryl-O_(n)C(O)—,    -   18) heterocyclyl-O_(n)C(O)—,    -   19) R⁸R⁹NC(═Y)—,    -   20) C₁-C₆ alkyl-S(O)₂—,    -   21) C₃-C₇ cycloalkyl-S(O)₂—,    -   22) aryl-S(O)₂—,    -   23) heteroaryl-S(O)₂—,    -   24) heterocyclyl-S(O)₂—,    -   25) fused aryl-C₃-C₇ cycloalkyl—,    -   26) fused heteroaryl-C₃-C₇ cycloalkyl—,    -   27) fused aryl-heterocyclyl—,    -   28) fused heteroraryl-heterocyclyl—,    -   29) fused aryl-C₃-C₇ cycloalkyl-O_(n)C(O)—,    -   30) fused heteroaryl-C₃-C₇ cycloalkyl-O_(n)C(O)—,    -   31) fused aryl-heterocyclyl-O_(n)C(O)—, or    -   32) fused heteroaryl-heterocyclyl-O_(n)C(O)—,        wherein the alkyl and the cycloalkyl are optionally substituted        with one or more R⁶ substituents, and the aryl, the heteroaryl        and the heterocyclyl are optionally substituted with one or more        R¹⁰ substituents;-   R⁶ is independently selected from:    -   1) halogen,    -   2) C₁-C₆ alkyl,    -   3) C₃-C₇ cycloalkyl,    -   4) haloalkyl,    -   5) aryl,    -   6) heteroaryl,    -   7) heterocyclyl,    -   8) OR⁷,    -   9) S(O)_(m)R⁷,    -   10) NR⁸R⁹,    -   11) COR⁷,    -   12) C(O)OR⁷,    -   13) OC(O)R⁷,    -   14) SC(O)R⁷,    -   15) CONR⁸R⁹,    -   16) S(O)₂NR⁸R⁹, or    -   17) N(═Y)NR⁸R⁹,        wherein the aryl, the heteroaryl and the heterocylyl are        optionally substituted with one or more R¹⁰ substituents;-   R⁷ is independently selected from:

1) H,

-   -   2) C₁-C₆ alkyl,    -   3) C₃-C₇ cycloalkyl,    -   4) haloalkyl,    -   5) aryl,    -   6) heteroaryl,    -   7) heterocyclyl,    -   8) C(═Y)NR⁸R⁹, or    -   9) C₁-C₆ alkyl-C₂-C₄ alkynyl,        wherein the aryl, heteroaryl, and heterocyclyl are optionally        substituted with one or more R¹⁰;

-   R³ and R⁹ are independently selected from:    -   1) H,    -   2) C₁-C₆ alkyl,    -   3) C₃-C₇ cycloalkyl,    -   4) haloalkyl,    -   5) aryl,    -   6) heteroaryl,    -   7) heterocyclyl,    -   8) COC₁-C₆ alkyl,    -   9) COC₃-C₃ cycloalkyl    -   10) CO-aryl,    -   11) CO-heteroaryl,    -   12) CO-heterocyclyl,    -   13) C(O)Y—C₁-C₆ alkyl,    -   14) C(O)Y—C₃-C₃ cycloalkyl    -   15) C(O)Y-aryl,    -   16) C(O)Y-heteroaryl, or    -   17) C(O)Y heterocyclyl,        wherein the aryl, the heteroaryl and the heterocyclyl are        optionally substituted with one or more R¹⁰ substituents;        or R⁸ and R⁹ together with the nitrogen atom to which they are        bonded form a five, six or seven membered heterocyclic ring        optionally substituted with one or more R⁶ substituents;

-   R¹⁰ is independently selected from:    -   1) halogen,    -   2) NO₂,    -   3) CN,    -   4) C₁-C₆ alkyl,    -   5) haloalkyl,    -   6) C₃-C₇ cycloalkyl,    -   7) OR⁷,    -   8) NR⁸R⁹,    -   9) SR⁷,    -   10) COR⁷,    -   11) CO₂R⁷,    -   12) S(O)_(m)R⁷    -   13) CONR⁸R⁹, or    -   14) S(O)₂NR⁸R⁹,        wherein the alkyl is optionally substituted with one or more R⁶        substituents;

-   R¹¹ is independently selected from    -   1) C₁-C₆ alkyl,    -   2) C₃-C₇ cycloalkyl,    -   3) aryl,    -   4) heteroaryl, or    -   5) heterocyclyl,        wherein the alkyl and the cycloalkyl are optionally substituted        with one or more R⁶ substituents, and the aryl, the heteroaryl        and the heterocyclyl are optionally substituted with one or more        R¹⁰ substituents;

-   R¹² is independently selected from    -   1) C₁-C₆ alkyl,    -   2) C₃-C₇ cycloalkyl,    -   3) haloalkyl,    -   4) aryl,    -   5) heteroaryl,    -   6) heterocyclyl,    -   7) C₁-C₆ alkyl-O_(n)C(O)—,    -   8) haloalkyl-O_(n)C(O)—,    -   9) C₃-C₇ cycloalkyl-O_(n)C(O)—,    -   10) aryl-O_(n)C(O)—,    -   11) heteroaryl-O_(n)C(O)—,    -   12) heterocyclyl-O_(n)C(O)—,    -   13) R⁸R⁹NC(O)—,    -   14) C₁-C₆ alkyl-S(O)_(m)—,    -   15) C₃-C₇ cycloalkyl-S(O)_(m)—,    -   16) aryl-S(O)_(m)—,    -   17) heteroaryl-S(O)_(m)—,    -   18) heterocyclyl-S(O)_(m)—,    -   19) fused aryl-C₃-C₇ cycloalkyl,    -   20) fused heteroaryl-C₃-C₇ cycloalkyl, or    -   21) C(═Y)—NR⁸R⁹,        wherein the alkyl and the cycloalkyl are optionally substituted        with one or more R⁶ substituents, and the aryl, the heteroaryl        and the heterocyclyl are optionally substituted with one or more        R¹⁰ substituents;

-   R¹³ is    -   1) H,    -   2) C₁-C₆ alkyl,    -   3) C₃-C₇ cycloalkyl,    -   4) haloalkyl,    -   5) aryl,    -   6) heteroaryl,    -   7) heterocyclyl,    -   8) C₁-C₆ alkyl-O_(n)C(O)—,    -   9) haloalkyl-O_(n)C(O)—,    -   10) C₃-C₇ cycloalkyl-O_(n)C(O)—,    -   11) aryl-O_(n)C(O)—,    -   12) heteroaryl-O_(n)C(O)—, or    -   13) heterocyclyl-O_(n)C(O)—;        or a prodrug, or a pharmaceutically acceptable salt, or labeled        with a detectable label or an affinity tag thereof.

If any variable, such as R⁶, R⁶⁰⁰, R¹⁰, R¹⁰⁰⁰ and the like, occurs morethan one time in any constituent structure, the definition of thevariable at each occurrence is independent at every other occurrence. Ifa substituent is itself substituted with one or more substituents, it isto be understood that that the one or more substituents may be attachedto the same carbon atom or different carbon atoms. Combinations ofsubstituents and variables defined herein are allowed only if theyproduce chemically stable compounds.

One skilled in the art will understand that substitution patterns andsubstituents on compounds of the present invention may be selected toprovide compounds that are chemically stable and can be readilysynthesized using the chemistry set forth in the examples and chemistrytechniques well known in the art using readily available startingmaterials.

It is to be understood that many substituents or groups described hereinhave functional group equivalents, which means that the group orsubstituent may be replaced by another group or substituent that hassimilar electronic, hybridization or bonding properties.

Definitions

Unless otherwise specified, the following definitions apply:

The singular forms “a”, “an” and “the” include corresponding pluralreferences unless the context clearly dictates otherwise.

As used herein, the term “comprising” is intended to mean that the listof elements following the word “comprising” are required or mandatorybut that other elements are optional and may or may not be present.

As used herein, the term “consisting of” is intended to mean includingand limited to whatever follows the phrase “consisting of”. Thus thephrase “consisting of” indicates that the listed elements are requiredor mandatory and that no other elements may be present.

As used herein, the term “alkyl” is intended to include both branchedand straight chain saturated aliphatic hydrocarbon groups having thespecified number of carbon atoms, for example, C₁-C₆ as in C₁-C₆-alkylis defined as including groups having 1, 2, 3, 4, 5 or 6 carbons in alinear or branched arrangement, and C₁-C₄ as in C₁-C₄ alkyl is definedas including groups having 1, 2, 3, or 4 carbons in a linear or branchedarrangement, and for example, C₁-C₂₀ as in C₁-C₂₀-alkyl is defined asincluding groups having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19 or 20 carbons in a linear or branched arrangement,Examples of C₁-C₆-alkyl and C₁-C₄ alkyl as defined above include, butare not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl,i-butyl, pentyl and hexyl.

As used herein, the term, “alkenyl” is intended to mean unsaturatedstraight or branched chain hydrocarbon groups having the specifiednumber of carbon atoms therein, and in which at least two of the carbonatoms are bonded to each other by a double bond, and having either E orZ regeochemistry and combinations thereof. For example, C₂-C₆ as inC₂-C₆ alkenyl is defined as including groups having 2, 3, 4, 5, or 6carbons in a linear or branched arrangement, at least two of the carbonatoms being bonded together by a double bond. Examples of C₂-C₆ alkenylinclude ethenyl (vinyl), 1-propenyl, 2-propenyl, 1-butenyl and the like.

As used herein, the term “alkynyl” is intended to mean unsaturated,straight chain hydrocarbon groups having the specified number of carbonatoms therein and in which at least two carbon atoms are bonded togetherby a triple bond. For example C₂-C₄ as in C₂-C₄ alkynyl is defined asincluding groups having 2, 3, or 4 carbon atoms in a chain, at least twoof the carbon atoms being bonded together by a triple bond. Examples ofsuch alkynyls include ethynyl, 1-propynyl, 2-propynyl and the like.

As used herein, the term “cycloalkyl” is intended to mean a monocyclicsaturated aliphatic hydrocarbon group having the specified number ofcarbon atoms therein, for example, C₃-C₇ as in C₃-C₇ cycloalkyl isdefined as including groups having 3, 4, 5, 6, or 7 carbons in amonocyclic arrangement. Examples of C₃-C₇ cycloalkyl as defined aboveinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl and cycloheptyl.

As used herein, the term “cycloalkenyl” is intended to mean a monocyclicsaturated aliphatic hydrocarbon group having the specified number ofcarbon atoms therein, for example, C₃-C₇ as in C₃-C₇ cycloalkenyl isdefined as including groups having 3, 4, 5, 6, or 7 carbons in amonocyclic arrangement. Examples of C₃-C₇ cycloalkenyl as defined aboveinclude, but are not limited to, cyclopentenyl, and cyclohexenyl.

As used herein, the term “halo” or “halogen” is intended to meanfluorine, chlorine, bromine and iodine.

As used herein, the term “haloalkyl” is intended to mean an alkyl asdefined above, in which each hydrogen atom may be successively replacedby a halogen atom. Examples of haloalkyls include, but are not limitedto, CH₂F, CHF₂ and CF₃.

As used herein, the term “aryl”, either alone or in combination withanother radical, means a carbocyclic aromatic monocyclic groupcontaining 6 carbon atoms which may be further fused to a second 5- or6-membered carbocyclic group which may be aromatic, saturated orunsaturated. Aryl includes, but is not limited to, phenyl, indanyl,1-naphthyl, 2-naphthyl and tetrahydronaphthyl. The aryls may beconnected to another group either at a suitable position on thecycloalkyl ring or the aromatic ring. For example:

Arrowed lines drawn from the ring system indicate that the bond may beattached to any of the suitable ring atoms.

As used herein, the term “biphenyl” is intended to mean two phenylgroups bonded together at any one of the available sites on the phenylring. For example:

As used herein, the term “heteroaryl” is intended to mean a monocyclicor bicyclic ring system of up to ten atoms, wherein at least one ring isaromatic, and contains from 1 to 4 hetero atoms selected from the groupconsisting of O, N, and S. The heteroaryl substituent may be attachedeither via a ring carbon atom or one of the heteroatoms. Examples ofheteroaryl groups include, but are not limited to thienyl,benzimidazolyl, benzo[b]thienyl, furyl, benzofuranyl, pyranyl,isobenzofuranyl, chromenyl, xanthenyl, 2H-pyrrolyl, pyrrolyl,imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl,indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl,4H-quinolizinyl, isoquinolyl, quinolyl, phthalazinyl, napthyridinyl,quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, isothiazolyl,isochromanyl, chromanyl, isoxazolyl, furazanyl, indolinyl, isoindolinyl,thiazolo[4,5-b]-pyridine, and fluoroscein derivatives such as

As used herein, the term “heterocycle”, “heterocyclic” or “heterocyclyl”is intended to mean a 5, 6, or 7 membered non-aromatic ring systemcontaining from 1 to 4 heteroatoms selected from the group consisting ofO, N and S. Examples of heterocycles include, but are not limited topyrrolidinyl, tetrahydrofuranyl, piperidyl, pyrrolinyl, piperazinyl,imidazolidinyl, morpholinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl,and

As used herein, the term “heterobicycle” either alone or in combinationwith another radical, is intended to mean a heterocycle as defined abovefused to another cycle, be it a heterocycle, an aryl or any other cycledefined herein. Examples of such heterobicycles include, but are notlimited to, coumarin, benzo[d][1,3]dioxole,2,3-dihydrobenzo[b][1,4]dioxine and3,4-dihydro-2H-benzo[b][1,4]dioxepine.

As used herein, the term “heteroaryl” is intended to mean a monocyclicor bicyclic ring system of up to ten atoms, wherein at least one ring isaromatic, and contains from 1 to 4 hetero atoms selected from the groupconsisting of O, N, and S. The heteroaryl substituent may be attachedeither via a ring carbon atom or one of the heteroatoms. Examples ofheteroaryl groups include, but are not limited to thienyl,benzimidazolyl, benzo[b]thienyl, furyl, benzofuranyl, pyranyl,isobenzofuranyl, chromenyl, xanthenyl, 2H-pyrrolyl, pyrrolyl,imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl,indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl,4H-quinolizinyl, isoquinolyl, quinolyl, phthalazinyl, napthyridinyl,quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, isothiazolyl,isochromanyl, chromanyl, isoxazolyl, furazanyl, indolinyl, andisoindolinyl,

As used herein, the term “heterocycle”, “heterocyclic” or “heterocyclyl”is intended to mean a 5, 6, or 7 membered non-aromatic ring systemcontaining from 1 to 4 heteroatoms selected from the group consisting ofO, N and S. Examples of heterocycles include, but are not limited topyrrolidinyl, tetrahydrofuranyl, piperidyl, pyrrolinyl, piperazinyl,imidazolidinyl, morpholinyl, imidazolinyl, pyrazolidinyl, andpyrazolinyl,

As used herein, the term “heteroatom” is intended to mean O, S or N.

As used herein, the term “actived diacid” is intended to mean a diacidwherein the carboxylic acid moieties have been transformed to, forexample, but not limited to, acid halides, a succinate esters, or HOBtesters, either in situ or in a separate synthetic step. For example,succinyl chloride and terephthaloyl chloride are examples of “diacidchlorides”. HOBt esters can be formed in situ by the treatment of adiacid with a dehydrating agent such as DCC, EDC, HBTU, or others, abase such as DIPEA, and HOBt in an appropriate solvent. The reaction ofan activated diacid with an amine will result in the conversion of theacid functionality to an amide functionality.

As used herein, the term “detectable label” is intended to mean a groupthat may be linked to a compound of the present invention to produce aprobe or to an IAP BIR domain, such that when the probe is associatedwith the BIR domain, the label allows either direct or indirectrecognition of the probe so that it may be detected, measured andquantified.

As used herein, the term “affinity tag” is intended to mean a ligand orgroup, which is linked to either a compound of the present invention orto an IAP BIR domain to allow another compound to be extracted from asolution to which the ligand or group is attached.

As used herein, the term “probe” is intended to mean a compound ofFormula I which is labeled with either a detectable label or an affinitytag, and which is capable of binding, either covalently ornon-covalently, to an IAP BIR domain. When, for example, the probe isnon-covalently bound, it may be displaced by a test compound. When, forexample, the probe is bound covalently, it may be used to formcross-linked adducts, which may be quantified and inhibited by a testcompound.

As used herein, the term “optionally substituted with one or moresubstituents” or its equivalent term “optionally substituted with atleast one substituent” is intended to mean that the subsequentlydescribed event of circumstances may or may not occur, and that thedescription includes instances where the event or circumstance occursand instances in which it does not. The definition is intended to meanfrom zero to five substituents.

If the substituents themselves are incompatible with the syntheticmethods of the present invention, the substituent may be protected witha suitable protecting group (PG) that is stable to the reactionconditions used in these methods. The protecting group may be removed ata suitable point in the reaction sequence of the method to provide adesired intermediate or target compound. Suitable protecting groups andthe methods for protecting and de-protecting different substituentsusing such suitable protecting groups are well known to those skilled inthe art; examples of which may be found in T. Greene and P. Wuts,Protecting Groups in Chemical Synthesis (3^(rd) ed.), John Wiley & Sons,NY (1999), which is incorporated herein by reference in its entirety.Examples of protecting groups used throughout include, but are notlimited to Fmoc, Bn, Boc, CBz and COCF₃. In some instances, asubstituent may be specifically selected to be reactive under thereaction conditions used in the methods of this invention. Under thesecircumstances, the reaction conditions convert the selected substituentinto another substituent that is either useful in an intermediatecompound in the methods of this invention or is a desired substituent ina target compound.

Abbreviations for α-amino acids used throughout are as follows:

Amino acid Abbreviation α-Amino butyric acid Abu Alanine Ala ArginineArg Aspartic acid Asp Asparagine Asn Cysteine Cys Glutamic acid GluGlutamine Gln Glycine Gly Isoleucine Ile Histidine His Leucine LeuLysine Lys Methionine Met Phenylalanine Phe Proline Pro Serine SerThreonine Thr Tryptophan Trp Tyrosine Tyr Valine Val

As used herein, the term “residue” when referring to α-amino acids isintended to mean a radical derived from the corresponding α-amino acidby eliminating the hydroxyl of the carboxy group and one hydrogen of theα-amino group. For example, the terms Gln, Ala, Gly, Ile, Arg, Asp, Phe,Ser, Leu, Cys, Asn, and Tyr represent the residues of L-glutamine,L-alanine, glycine, L-isoleucine, L-arginine, L-aspartic acid,L-phenylalanine, L-serine, L-leucine, L-cysteine, L-asparagine, andL-tyrosine, respectively.

As used herein, the term “subject” is intended to mean humans andnon-human mammals such as primates, cats, dogs, swine, cattle, sheep,goats, horses, rabbits, rats, mice and the like.

As used herein, the term “prodrug” is intended to mean a compound thatmay be converted under physiological conditions or by solvolysis to abiologically active compound of the present invention. Thus, the term“prodrug” refers to a precursor of a compound of the invention that ispharmaceutically acceptable. A prodrug may be inactive or displaylimited activity when administered to a subject in need thereof, but isconverted in vivo to an active compound of the present invention.Typically, prodrugs are transformed in vivo to yield the compound of theinvention, for example, by hydrolysis in blood or other organs byenzymatic processing. The prodrug compound often offers advantages ofsolubility, tissue compatibility or delayed release in the subject (see,Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier,Amsterdam). The definition of prodrug includes any covalently bondedcarriers which release the active compound of the invention in vivo whensuch prodrug is administered to a subject. Prodrugs of a compound of thepresent invention may be prepared by modifying functional groups presentin the compound of the invention in such a way that the modificationsare cleaved, either in routine manipulation or in vivo, to a parentcompound of the invention.

As used herein, the term “pharmaceutically acceptable carrier, diluentor excipient” is intended to mean, without limitation, any adjuvant,carrier, excipient, glidant, sweetening agent, diluent, preservative,dye/colorant, flavor enhancer, surfactant, wetting agent, dispersingagent, suspending agent, stabilizer, isotonic agent, solvent,emulsifier, or encapsulating agent, such as a liposome, cyclodextrins,encapsulating polymeric delivery systems or polyethyleneglycol matrix,which is acceptable for use in the subject, preferably humans.

As used herein, the term “pharmaceutically acceptable salt” is intendedto mean both acid and base addition salts.

As used herein, the term “pharmaceutically acceptable acid additionsalt” is intended to mean those salts which retain the biologicaleffectiveness and properties of the free bases, which are notbiologically or otherwise undesirable, and which are formed withinorganic acids such as hydrochloric acid, hydrobromic acid, sulfuricacid, nitric acid, phosphoric acid and the like, and organic acids suchas acetic acid, trifluoroacetic acid, propionic acid, glycolic acid,pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid,fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid,mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid, and the like.

As used herein, the term “pharmaceutically acceptable base additionsalt” is intended to mean those salts which retain the biologicaleffectiveness and properties of the free acids, which are notbiologically or otherwise undesirable. These salts are prepared fromaddition of an inorganic base or an organic base to the free acid. Saltsderived from inorganic bases include, but are not limited to, thesodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc,copper, manganese, aluminum salts and the like. Salts derived fromorganic bases include, but are not limited to, salts of primary,secondary, and tertiary amines, substituted amines including naturallyoccurring substituted amines, cyclic amines and basic ion exchangeresins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine,caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine,glucosamine, methylglucamine, theobromine, purines, piperazine,piperidine, N-ethylpiperidine, polyamine resins and the like.

As used herein, the term “BIR domain binding” is intended to mean theaction of a compound of the present invention upon an IAP BIR domain,which blocks or diminishes the binding of IAPs to BIR binding proteinsor is involved in displacing BIR binding proteins from an IAP. Examplesof BIR binding proteins include, but are not limited to, caspases andmitochondrially derived BIR binding proteins such as Smac, OmiNWTR2A andthe like.

As used herein, the term “insufficient apoptosis” is intended to mean astate wherein a disease is caused or continues because cells deleteriousto the subject have not apoptosed. This includes, but is not limited to,cancer cells that survive in a subject without treatment, cancer cellsthat survive in a subject during or following anti-cancer treatment, orimmune cells whose action is deleterious to the subject, and includes,neutrophils, monocytes and auto-reactive T-cells.

As used herein, the term “therapeutically effective amount” is intendedto mean an amount of a compound of Formula I or II which, whenadministered to a subject is sufficient to effect treatment for adisease-state associated with insufficient apoptosis. The amount of thecompound of Formula I will vary depending on the compound, the conditionand its severity, and the age of the subject to be treated, but can bedetermined routinely by one of ordinary skill in the art having regardto his own knowledge and to this disclosure.

As used herein, the term “treating” or “treatment” is intended to meantreatment of a disease-state associated with insufficient apoptosis, asdisclosed herein, in a subject, and includes: (i) preventing a diseaseor condition associated with insufficient apoptosis from occurring in asubject, in particular, when such mammal is predisposed to the diseaseor condition but has not yet been diagnosed as having it; (ii)inhibiting a disease or condition associated with insufficientapoptosis, i.e., arresting its development; or (iii) relieving a diseaseor condition associated with insufficient apoptosis, i.e., causingregression of the condition.

As used herein, the term “treating cancer” is intended to mean theadministration of a pharmaceutical composition of the present inventionto a subject, preferably a human, which is afflicted with cancer tocause an alleviation of the cancer by killing, inhibiting the growth, orinhibiting the metastasis of the cancer cells.

As used herein, the term “preventing disease” is intended to mean, inthe case of cancer, the post-surgical, post-chemotherapy orpost-radiotherapy administration of a pharmaceutical composition of thepresent invention to a subject, preferably a human, which was afflictedwith cancer to prevent the regrowth of the cancer by killing, inhibitingthe growth, or inhibiting the metastasis of any remaining cancer cells.Also included in this definition is the prevention of prosurvivalconditions that lead to diseases such as asthma, MS and the like.

As used herein, the term “synergistic effect” is intended to mean thatthe effect achieved with the combination of the compounds of the presentinvention and either the chemotherapeutic agents or death receptoragonists of the invention is greater than the effect which is obtainedwith only one of the compounds, agents or agonists, or advantageouslythe effect which is obtained with the combination of the abovecompounds, agents or agonists is greater than the addition of theeffects obtained with each of the compounds, agents or agonists usedseparately. Such synergy enables smaller doses to be given.

As used herein, the term “apoptosis” or “programmed cell death” isintended to mean the regulated process of cell death wherein a dyingcell displays a set of well-characterized biochemical hallmarks thatinclude cell membrane blebbing, cell soma shrinkage, chromatincondensation, and DNA laddering, as well as any caspase-mediated celldeath.

As used herein, the term “BIR domain” or “BIR” are used interchangeablythroughout and are intended to mean a domain which is characterized by anumber of invariant amino acid residue including conserved cysteines andone conserved hisitidine residue within the sequenceCys-(Xaa1)₂Cys-(Xaa1)₁₆His-(Xaa1)₆₋₈Cys. Typically, the amino acidsequence of the consensus sequence is:Xaa1-Xaa1-Xaa1-Arg-Leu-Xaa1-Thr-Phe-Xaa1-Xaa1-Trp-Pro-Xaa2-Xaa1-Xaa1-Xaa2-Xaa2-Xaa1-Xaa1-Xaa1-Xaa1-Leu-Ala-Xaa1-Ala-Gly-Phe-Tyr-Tyr-Xaa1-Gly-Xaa1-Xaa1-Asp-Xaa1-Val-Xaa1-Cys-Phe-Xaa1-Cys-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Trp-Xaa1-Xaa1-Xaa1-Asp-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-His-Xaa1-Xaa1-Xaa1-Xaa1-Pro-Xaa1-Cys-Xaa1-Phe-Val,wherein Xaa1 is any amino acid and Xaa2 is any amino acid or is absent.Preferably the sequence is substantially identical to one of the BIRdomain sequences provided for XIAP, HIAP1, or HIAP2 herein.

The BIR domain residues are listed below (see Genome Biology (2001)1-10):

XIAP HIAP-1 HIAP-2 BIR1 21-93  41-113 24-96 BIR2 159-230 179-250 164-235BIR3 258-330 264-336 250-322 Seq. # P98170 XP-006266 XP-006267

As used herein, the term “ring zinc finger” or “RZF” is intended to meana domain having the amino acid sequence of the consensus sequence:Glu-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa-1-Xaa2-Xaa1-Xaa1-Xaa1-Cys-Lys-Xaa3-Cys-Met-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa3-X-aa1-Phe-Xaa1-Pro-Cys-Gly-His-Xaa1-Xaa1-Xaa1-Cys-Xaa1-Xaa1-Cys-Ala-Xaa1-Xaa-1-Xaa1-Xaa1-Xaa1-Cys-Pro-Xaa1-Cys,wherein Xaa1 is any amino acid, Xaa2 is Glu or Asp, and Xaa3 is Val orIle.

As used herein, the term “IAP” is intended to mean a polypeptide orprotein, or fragment thereof, encoded by an IAP gene. Examples of IAPsinclude, but are not limited to human or mouse NAIP (Birc 1), HIAP-1(cIAP2, Birc 3), HIAP-2 (cIAP1, Birc 2), XIAP (Birc 4), survivin (Birc5), livin (ML-IAP, Birc 7), ILP-2 (Birc 8) and Apollon/BRUCE (Birc 6)(see for example US Patent Numbers 6,107,041; 6,133,437; 6,156,535;6,541,457; 6,656,704; 6,689,562; Deveraux and Reed, Genes Dev. 13,239-252,1999; Kasof and Gomes, J. Biol. Chem., 276, 3238-3246, 2001;Vucic et al., Curr. Biol. 10, 1359-1366, 2000; Ashab et al. FEBS Lett.,495, 56-60, 2001, the contents of which are hereby incorporated byreference).

As used herein, the term “IAP gene” is intended to mean a gene encodinga polypeptide having at least one BIR domain and which is capable ofmodulating (inhibiting or enhancing) apoptosis in a cell or tissue. TheIAP gene is a gene having about 50% or greater nucleotide sequenceidentity to at least one of human or mouse NAIP (Birc 1), HIAP-1 (cIAP2,Birc 3), HIAP-2 (cIAP1, Birc 2), XIAP (Birc 4), survivin (Birc 5), livin(ML-IAP, Birc 7), ILP-2 (Birc 8) and Apollon/BRUCE (Birc 6). The regionof sequence over which identity is measured is a region encoding atleast one BIR domain and a ring zinc finger domain. Mammalian IAP genesinclude nucleotide sequences isolated from any mammalian source.

As used herein, the term “IC₅₀” is intended to mean an amount,concentration or dosage of a particular compound of the presentinvention that achieves a 50% inhibition of a maximal response, such asdisplacement of maximal fluorescent probe binding in an assay thatmeasures such response.

As used herein, the term “EC₅₀” is intended to mean an amount,concentration or dosage of a particular compound of the presentinvention that achieves a 50% inhibition of cell survival.

As used herein, the term “modulate” or “modulating” is intended to meanthe treatment, prevention, suppression, enhancement or induction of afunction or condition using the compounds of the present invention. Forexample, the compounds of the present invention can modulate IAPfunction in a subject, thereby enhancing apoptosis by significantlyreducing, or essentially eliminating the interaction of activatedapoptotic proteins, such as caspase-3, 7 and 9, with the BIR domains ofmammalian IAPs or by inducing the loss of XIAP protein in a cell.

As used herein, the term “enhancing apoptosis” is intended to meanincreasing the number of cells that apoptose in a given cell populationeither in vitro or in vivo. Examples of cell populations include, butare not limited to, ovarian cancer cells, colon cancer cells, breastcancer cells, lung cancer cells, pancreatic cancer cells, or T cells andthe like. It will be appreciated that the degree of apoptosisenhancement provided by an apoptosis-enhancing compound of the presentinvention in a given assay will vary, but that one skilled in the artcan determine the statistically significant change in the level ofapoptosis that identifies a compound that enhances apoptosis otherwiselimited by an IAP. Preferably “enhancing apoptosis” means that theincrease in the number of cells undergoing apoptosis is at least 25%,more preferably the increase is 50%, and most preferably the increase isat least one-fold. Preferably the sample monitored is a sample of cellsthat normally undergo insufficient apoptosis (i.e., cancer cells).Methods for detecting the changes in the level of apoptosis (i.e.,enhancement or reduction) are described in the Examples and includemethods that quantitate the fragmentation of DNA, methods thatquantitate the translocation phosphatoylserine from the cytoplasmic tothe extracellular side of the membrane, determination of activation ofthe caspases and methods quantitate the release of cytochrome C and theapoptosis inhibitory factor into the cytoplasm by mitochondria.

As used herein, the term “proliferative disease” or “proliferativedisorder” is intended to mean a disease that is caused by or results ininappropriately high levels of cell division, inappropriately low levelsof apoptosis, or both. For example, cancers such as lymphoma, leukemia,melanoma, ovarian cancer, breast cancer, pancreatic cancer, and lungcancer, and autoimmune disorders are all examples of proliferativediseases.

As used herein, the term “death receptor agonist” is intended to mean anagent capable of stimulating by direct or indirect contact the proapoptotic response mediated by the death-receptors. For example, anagonist TRAIL receptor Antibody would bind to TRAIL receptor (S) andtrigger an apoptotic response. On the other hand, other agent such asinterferon-a could trigger the release of endogeneous TRAIL and/or upregulate the TRAIL receptors in such a way that the cell pro-apoptoticresponse is amplified.

The compounds of the present invention, or their pharmaceuticallyacceptable salts may contain one or more asymmetric centers, chiral axesand chiral planes and may thus give rise to enantiomers, diastereomers,and other stereoisomeric forms and may be defined in terms of absolutestereochemistry, such as (R)— or (S)— or, as (D)- or (L)- for aminoacids. The present invention is intended to include all such possibleisomers, as well as, their racemic and optically pure forms. Opticallyactive (+) and (−), (R)— and (S)—, or (D)- and (L)-isomers may beprepared using chiral synthons or chiral reagents, or resolved usingconventional techniques, such as reverse phase HPLC. The racemicmixtures may be prepared and thereafter separated into individualoptical isomers or these optical isomers may be prepared by chiralsynthesis. The enantiomers may be resolved by methods known to thoseskilled in the art, for example by formation of diastereoisomeric saltswhich may then be separated by crystallization, gas-liquid or liquidchromatography, selective reaction of one enantiomer with an enantiomerspecific reagent. It will also be appreciated by those skilled in theart that where the desired enantiomer is converted into another chemicalentity by a separation technique, an additional step is then required toform the desired enantiomeric form. Alternatively specific enantiomersmay be synthesized by asymmetric synthesis using optically activereagents, substrates, catalysts, or solvents or by converting oneenantiomer to another by asymmetric transformation.

Certain compounds of the present invention may exist in Zwitterionicform and the present invention includes Zwitterionic forms of thesecompounds and mixtures thereof.

Utilities

The compounds of the present invention are useful as IAP BIR domainbinding compounds and as such the compounds, compositions and method ofthe present invention include application to the cells or subjectsafflicted with or having a predisposition towards developing aparticular disease state, which is characterized by insufficientapoptosis. Thus, the compounds, compositions and methods of the presentinvention are used to treat cellular proliferative diseases/disorders,which include, but are not limited to, i) cancer, ii) autoimmunedisease, iii) inflammatory disorders, iv) proliferation induced postmedical procedures, including, but not limited to, surgery, angioplasty,and the like.

The compounds of the present invention may also be useful in thetreatment of diseases in which there is a defect in the programmedcell-death or the apoptotic machinery (TRAIL, FAS, apoptosome), such asmultiple sclerosis, artherosclerosis, inflammation, autoimmunity and thelike.

The treatment involves administration to a subject in need thereof acompound of the present invention or a pharmaceutically acceptable saltthereof, or a pharmaceutical composition comprising a pharmaceuticalcarrier and a therapeutically effective amount of a compound of thepresent invention, or a pharmaceutically acceptable salt thereof. Inparticular, the compounds, compositions and methods of the presentinvention are useful for the treatment of cancer including solid tumorssuch as skin, breast, brain, lung, testicular carcinomas, and the like.Cancers that may be treated by the compounds, compositions and methodsof the invention include, but are not limited to the following:

Tissue Example Adrenal gland neuroblastoma Bone osteogenic sarcoma(osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma,chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cellsarcoma), multiple myeloma, malignant giant cell tumor chordoma,osteochronfroma (osteocartilaginous exostoses), benign chondroma,chondroblastoma, chondromyxofibroma, osteoid osteoma and giant celltumors Cardiac sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma,liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratomaGastrointestinal esophagus (squamous cell carcinoma, adenocarcinoma,leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma,leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma,glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel(adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma)Genitourinary kidney (adenocarcinoma, Wilm's tumor [nephroblastoma],tract lymphoma, leukemia), bladder and urethra (squamous cell carcinoma,transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma,sarcoma), testis (seminoma, teratoma, embryonal carcinoma,teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma,fibroma, fibroadenoma, adenomatoid tumors, lipoma) Gynecological uterus(endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervicaldysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma,mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecalcell tumors, Sertoli- Leydig cell tumors, dysgerminoma, malignantteratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma,adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma,squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma),fallopian tubes (carcinoma) Hematologic blood (myeloid leukemia [acuteand chronic], acute lymphoblastic leukemia, chronic lymphocyticleukemia, myeloproliferative diseases, multiple myeloma, myelodysplasticsyndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignantlymphoma] Liver hepatoma (hepatocellular carcinoma), cholangiocarcinoma,hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma Lungbronchogenic carcinoma (squamous cell, undifferentiated small cell,undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar)carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatoushamartoma, mesothelioma Nervous system skull (osteoma, hemangioma,granuloma, xanthoma, osteitis deformans), meninges (meningioma,meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma,glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform,oligodendroglioma, schwannoma, retinoblastoma, congenital tumors),spinal cord neurofibroma, meningioma, glioma, sarcoma) Skin malignantmelanoma, basal cell carcinoma, squamous cell carcinoma, Karposi'ssarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids

The compounds of the present invention, or their pharmaceuticallyacceptable salts or their prodrugs, may be administered in pure form orin an appropriate pharmaceutical composition, and can be carried out viaany of the accepted modes of Galenic pharmaceutical practice.

The pharmaceutical compositions of the present invention can be preparedby mixing a compound of the present invention with an appropriatepharmaceutically acceptable carrier, diluent or excipient, and may beformulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants, gels, microspheres, andaerosols. Typical routes of administering such pharmaceuticalcompositions include, without limitation, oral, topical, transdermal,inhalation, parenteral (subcutaneous injections, intravenous,intramuscular, intrasternal injection or infusion techniques),sublingual, ocular, rectal, vaginal, and intranasal. Pharmaceuticalcompositions of the present invention are formulated so as to allow theactive ingredients contained therein to be bioavailable uponadministration of the composition to a subject. Compositions that willbe administered to a subject or patient take the form of one or moredosage units, where for example, a tablet may be a single dosage unit,and a container of a compound of the present invention in aerosol formmay hold a plurality of dosage units. Actual methods of preparing suchdosage forms are known, or will be apparent, to those skilled in thisart; for example, see Remington's Pharmaceutical Sciences, 18th Ed.,(Mack Publishing Company, Easton, Pa., 1990). The composition to beadministered will, in any event, contain a therapeutically effectiveamount of a compound of the present invention, or a pharmaceuticallyacceptable salt thereof, for treatment of a disease-state as describedabove.

A pharmaceutical composition of the present invention may be in the formof a solid or liquid. In one aspect, the carrier(s) are particulate, sothat the compositions are, for example, in tablet or powder form. Thecarrier(s) may be liquid, with the compositions being, for example, anoral syrup, injectable liquid or an aerosol, which is useful in, forexample inhalatory administration.

For oral administration, the pharmaceutical composition is preferably ineither solid or liquid form, where semi-solid, semi-liquid, suspensionand gel forms are included within the forms considered herein as eithersolid or liquid.

As a solid composition for oral administration, the pharmaceuticalcomposition may be formulated into a powder, granule, compressed tablet,pill, capsule, chewing gum, wafer or the like form. Such a solidcomposition will typically contain one or more inert diluents or ediblecarriers. In addition, one or more of the following may be present:binders such as carboxymethylcellulose, ethyl cellulose,microcrystalline cellulose, gum tragacanth or gelatin; excipients suchas starch, lactose or dextrins, disintegrating agents such as alginicacid, sodium alginate, Primogel, corn starch and the like; lubricantssuch as magnesium stearate or Sterotex; glidants such as colloidalsilicon dioxide; sweetening agents such as sucrose or saccharin; aflavoring agent such as peppermint, methyl salicylate or orangeflavoring; and a coloring agent.

When the pharmaceutical composition is in the form of a capsule, e.g., agelatin capsule, it may contain, in addition to materials of the abovetype, a liquid carrier such as polyethylene glycol or oil such assoybean or vegetable oil.

The pharmaceutical composition may be in the form of a liquid, e.g., anelixir, syrup, solution, emulsion or suspension. The liquid may be fororal administration or for delivery by injection, as two examples. Whenintended for oral administration, preferred composition contain, inaddition to the present compounds, one or more of a sweetening agent,preservatives, dye/colorant and flavor enhancer. In a compositionintended to be administered by injection, one or more of a surfactant,preservative, wetting agent, dispersing agent, suspending agent, buffer,stabilizer and isotonic agent may be included.

The liquid pharmaceutical compositions of the present invention, whetherthey be solutions, suspensions or other like form, may include one ormore of the following adjuvants: sterile diluents such as water forinjection, saline solution, preferably physiological saline, Ringer'ssolution, isotonic sodium chloride, fixed oils such as synthetic mono ordiglycerides which may serve as the solvent or suspending medium,polyethylene glycols, glycerin, propylene glycol or other solvents;antibacterial agents such as benzyl alcohol or methyl paraben;encapsulating agents such as cyclodextrins or functionalizedcyclodextrins, including, but not limited to, α, β, orδ-hydroxypropylcyclodextins or Captisol; antioxidants such as ascorbicacid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates andagents for the adjustment of tonicity such as sodium chloride ordextrose. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic. Aninjectable pharmaceutical composition is preferably sterile.

A liquid pharmaceutical composition of the present invention used foreither parenteral or oral administration should contain an amount of acompound of the present invention such that a suitable dosage will beobtained. Typically, this amount is at least 0.01% of a compound of thepresent invention in the composition. When intended for oraladministration, this amount may be varied to be between 0.1 and about70% of the weight of the composition. For parenteral usage, compositionsand preparations according to the present invention are prepared so thata parenteral dosage unit contains between 0.01 to 10% by weight of thecompound of the present invention. Pharmaceutical compositions may befurther diluted at the time of administration; for example a parenteralformulation may be further diluted with a sterile, isotonic solution forinjection such as 0.9% saline, 5 wt % dextrose (D5W), Ringer's solution,or others.

The pharmaceutical composition of the present invention may be used fortopical administration, in which case the carrier may suitably comprisea solution, emulsion, ointment or gel base. The base, for example, maycomprise one or more of the following: petrolatum, lanolin, polyethyleneglycols, bee wax, mineral oil, diluents such as water and alcohol, andemulsifiers and stabilizers. Thickening agents may be present in apharmaceutical composition for topical administration. If intended fortransdermal administration, the composition may include a transdermalpatch or iontophoresis device. Topical formulations may contain aconcentration of the compound of the present invention from about 0.1 toabout 10% w/v (weight per unit volume).

The pharmaceutical composition of the present invention may be used forrectal administration to treat for example, colon cancer, in the form,e.g., of a suppository, which will melt in the rectum and release thedrug. The composition for rectal administration may contain anoleaginous base as a suitable nonirritating excipient. Such basesinclude, without limitation, lanolin, cocoa butter and polyethyleneglycol.

The pharmaceutical composition of the present invention may includevarious materials, which modify the physical form of a solid or liquiddosage unit. For example, the composition may include materials thatform a coating shell around the active ingredients. The materials thatform the coating shell are typically inert, and may be selected from,for example, sugar, shellac, and other enteric coating agents.Alternatively, the active ingredients may be encased in a gelatincapsule.

The pharmaceutical composition of the present invention in solid orliquid form may include an agent that binds to the compound of thepresent invention and thereby assists in the delivery of the compound.Suitable agents that may act in this capacity include, but are notlimited to, a monoclonal or polyclonal antibody, a protein or aliposome.

The pharmaceutical composition of the present invention may consist ofdosage units that can be administered as an aerosol. The term aerosol isused to denote a variety of systems ranging from those of colloidalnature to systems consisting of pressurized packages. Delivery may be bya liquefied or compressed gas or by a suitable pump system thatdispenses the active ingredients. Aerosols of compounds of the presentinvention may be delivered in single phase, bi-phasic, or tri-phasicsystems in order to deliver the active ingredient(s). Delivery of theaerosol includes the necessary container, activators, valves,subcontainers, and the like, which together may form a kit. One skilledin the art, without undue experimentation may determine preferredaerosols.

The pharmaceutical compositions of the present invention may be preparedby methodology well known in the pharmaceutical art. For example, apharmaceutical composition intended to be administered by injection canbe prepared by admixing a compound of the present invention withsterile, distilled water so as to form a solution. A surfactant may beadded to facilitate the formation of a homogeneous solution orsuspension. Surfactants are compounds that non-covalently interact withthe compound of the present invention so as to facilitate dissolution orhomogeneous suspension of the compound in the aqueous delivery system.

The compounds of the present invention, or their pharmaceuticallyacceptable salts, are administered in a therapeutically effectiveamount, which will vary depending upon a variety of factors includingthe activity of the specific compound employed; the metabolic stabilityand length of action of the compound; the age, body weight, generalhealth, sex, and diet of the patient; the mode and time ofadministration; the rate of excretion; the drug combination; theseverity of the particular disorder or condition; and the subjectundergoing therapy. Generally, a therapeutically effective daily dosemay be from about 0.1 mg to about 40 mg/kg of body weight per day ortwice per day of a compound of the present invention, or apharmaceutically acceptable salt thereof.

Combination Therapy

The compounds of the present invention, or pharmaceutically acceptablesalts thereof, may also be administered simultaneously with, prior to,or after administration of one or more of the therapeutic agentsdescribed below. Such combination therapy may include administration ofa single pharmaceutical dosage formulation which contains a compound ofthe present invention and one or more additional agents given below, aswell as administration of the compound of the present invention and eachof additional agent in its own separate pharmaceutical dosageformulation. For example, a compound of the present invention and achemotherapeutic agent, such as taxol (paclitaxel), taxotere, etoposide,cisplatin, vincristine, vinblastine, and the like, can be administeredto the patient either together in a single oral dosage composition suchas a tablet or capsule, or each agent administered in separate oraldosage formulations or via intravenous injection. Where separate dosageformulations are used, the compounds of the present invention and one ormore additional agents can be administered at essentially the same time,i.e., concurrently, or at separately staggered times, i.e.,sequentially; combination therapy is understood to include all theseregimens. In addition, these compounds may synergize with molecules thatmay stimulate the death receptor apoptotic pathway through a direct orindirect manner, as for example, the compounds of the present inventionmay be used in combination with soluble TRAIL any agent or proceduresthat can cause an increase in circulating level of TRAIL, such asinterferon-alpha or radiation.

Thus, the present invention also encompasses the use of the compounds ofthe present invention in combination with radiation therapy or one ormore additional agents such as those described in WO 03/099211(PCT/US03/15861), which is hereby incorporated by reference.

Examples of such additional agents include, but are not limited to thefollowing:

-   a) an estrogen receptor modulator,-   b) an androgen receptor modulator,-   c) retinoid receptor modulator,-   d) a cytotoxic agent,-   e) an antiproliferative agent,-   f) a prenyl-protein transferase inhibitor,-   g) an HMG-CoA reductase inhibitor,-   h) an HIV protease inhibitor,-   i) a reverse transcriptase inhibitor,-   k) an angiogenesis inhibitor,-   l) a PPAR-γ agonist,-   m) a PPAR-δ agonist,-   n) an inhibitor of inherent multidrug resistance,-   o) an anti-emetic agent,-   p) an agent useful in the treatment of anemia,-   q) agents useful in the treatment of neutropenia,-   r) an immunologic-enhancing drug,-   s) a proteasome inhibitor such as Velcade and MG132    (7-Leu-Leu-aldehyde) (see He at al. in Oncogene (2004) 23,    2554-2558),-   t) an HDAC inhibitor, such as sodium butyrate, phenyl butyrate,    hydroamic acids, cyclin tetrapeptide and the like (see Rosato et    al., Molecular Cancer Therapeutics 2003, 1273-1284),-   u) an inhibitor of the chymotrypsin-like activity in the proteasome,-   v) E3 ligase inhibitors,-   w) a modulator of the immune system such as interferon-alpha and    ionizing radition (UVB) that can induce the release of cytokines,    such as the interleukins, TNF, or induce release of Death receptor    Ligands such as TRAIL,-   x) a modulator of death receptors TRAIL and TRAIL receptor agonists    such as the humanized antibodies HGS-ETR1 and HGS-ETR2,    or in combination or sequentially with radiation therapy, so as to    treat the cancer.

Additional combinations may also include agents which reduce thetoxicity of the aforesaid agents, such as hepatic toxicity, neuronaltoxicity, nephprotoxicity and the like.

In one example, co-administration of one of the compounds of Formula Iof the present invention with a death receptor agonist such as TRAIL,such as a small molecule or an antibody that mimics TRAIL may cause anadvantageous synergistic effect. Moreover, the compounds of the presentinvention may be used in combination with any compounds that cause anincrease in circulating levels of TRAIL.

Vinca Alkaloids and Related Compounds

Vinca alkaloids that can be used in combination with the nucleobaseoligomers of the invention to treat cancer and other neoplasms includevincristine, vinblastine, vindesine, vinflunine, vinorelbine, andanhydrovinblastine.

Dolastatins are oligopeptides that primarily interfere with tubulin atthe vinca alkaloid binding domain. These compounds can also be used incombination with the compounds of the invention to treat cancer andother neoplasms. Dolastatins include dolastatin-10 (NCS 376128),dolastatin-15, ILX651, TZT-1027, symplostatin 1, symplostatin 3, andLU103793 (cemadotin).

Cryptophycins (e.g., cryptophycin 1 and cryptophycin 52 (LY355703)) bindtubulin within the vinca alkaloid-binding domain and induce G2/M arrestand apoptosis. Any of these compounds can be used in combination withthe compounds of the invention to treat cancer and other neoplasms.

Other microtubule disrupting compounds that can be used in conjunctionwith the compounds of the invention to treat cancer and other neoplasmsare described in U.S. Pat. Nos. 6,458,765; 6,433,187; 6,323,315;6,258,841; 6,143,721; 6,127,377; 6,103,698; 6,023,626; 5,985,837;5,965,537; 5,955,423; 5,952,298; 5,939,527; 5,886,025; 5,831,002;5,741,892; 5,665,860; 5,654,399; 5,635,483; 5,599,902; 5,530,097;5,521,284; 5,504,191; 4,879,278; and 4,816,444, and U.S. patentapplication Publication Nos. 2003/0153505 A1; 2003/0083263 A1; and2003/0055002 A1, each of which is hereby incorporated by reference.

Taxanes and Other Micortubule Stabilizing Compounds

Taxanes such as paclitaxel, doxetaxel, RPR 109881A, SB-T-1213,SB-T-1250, SB-T-101187, BMS-275183, BRT 216, DJ-927, MAC-321, IDN5109,and IDN5390 can be used in combination with the compounds of theinvention to treat cancer and other neoplasms. Taxane analogs (e.g.,BMS-184476, BMS-188797) and functionally related non-taxanes (e.g.,epothilones (e.g., epothilone A, epothilone B (EP0906), deoxyepothiloneB, and epothilone B lactam (BMS-247550)), eleutherobin, discodermolide,2-epi-discodermolide, 2-des-methyldiscodermolide,5-hydroxymethyldiscoder-molide, 19-des-aminocarbonyldiscodermolide,9(13)-cyclodiscodermolide, and laulimalide) can also be used in themethods and compositions of the invention.

Other microtubule stabilizing compounds that can be used in combinationwith the compounds of the invention to treat cancer and other neoplasmsare described in U.S. Pat. Nos. 6,624,317; 6,610,736; 6,605,599;6,589,968; 6,583,290; 6,576,658; 6,515,017; 6,531,497; 6,500,858;6,498,257; 6,495,594; 6,489,314; 6,458,976; 6,441,186; 6,441,025;6,414,015; 6,387,927; 6,380,395; 6,380,394; 6,362,217; 6,359,140;6,306,893; 6,302,838; 6,300,355; 6,291,690; 6,291,684; 6,268,381;6,262,107; 6,262,094; 6,147,234; 6,136,808; 6,127,406; 6,100,411;6,096,909; 6,025,385; 6,011,056; 5,965,718; 5,955,489; 5,919,815;5,912,263; 5,840,750; 5,821,263; 5,767,297; 5,728,725; 5,721,268;5,719,177; 5,714,513; 5,587,489; 5,473,057; 5,407,674; 5,250,722;5,010,099; and 4,939,168; and U.S. patent application Publication Nos.2003/0186965 A1; 2003/0176710 A1; 2003/0176473 A1; 2003/0144523 A1;2003/0134883 A1; 2003/0087888 A1; 2003/0060623 A1; 2003/0045711 A1;2003/0023082 A1; 2002/0198256 A1; 2002/0193361 A1; 2002/0188014 A1;2002/0165257 A1; 2002/0156110 A1; 2002/0128471 A1; 2002/0045609 A1;2002/0022651 A1; 2002/0016356 A1; 2002/0002292 A1, each of which ishereby incorporated by reference.

Other chemotherapeutic agents that may be administered with a compoundof the present invention are listed in the following Table:

Alkylating cyclophosphamide mechlorethamine agents lomustine thiotepabusulfan streptozocin procarbazine chlorambucil ifosfamide temozolomidealtretamine dacarbazine melphalan semustine estramustine phosphatecarmustine hexamethylmelamine Platinum agents cisplatin tetraplatincarboplatinum BBR-3464 (Hoffmann-La Roche) oxaliplatin OrmiplatinZD-0473 (AnorMED) SM-11355 (Sumitomo) spiroplatinum iproplatinlobaplatin (Aeterna) AP-5280 (Access) carboxyphthalatoplatinumsatraplatin (Johnson Matthey) Antimetabolites azacytidine6-mercaptopurine tomudex hydroxyurea gemcitabine 6-thioguaninetrimetrexate decitabine (SuperGen) capecitabine cytarabindeoxycoformycin clofarabine (Bioenvision) 5-fluorouracil 2-fluorodeoxyfludarabine cytidine floxuridine irofulven (MGI Pharma) methotrexatepentostatin DMDC (Hoffmann-La Roche) 2-chlorodeoxyadenosine idatrexateraltitrexed ethynylcytidine (Taiho) Topoisomerase amsacrine TAS-103(Taiho) inhibitors rubitecan (SuperGen) Topotecan epirubicinelsamitrucin (Spectrum) dexrazoxanet exatecan mesylate (Daiichi)(TopoTarget) etoposide J-107088 (Merck & Co) quinamed (ChemGenex)pixantrone (Novuspharma) teniposide or mitoxantrone BNP-1350(BioNumerik) gimatecan (Sigma-Tau) rebeccamycin analogue (Exelixis)irinotecan (CPT-11) CKD-602 (Chong Kun Dang) diflomotecan(Beaufour-Ipsen) BBR-3576 (Novuspharma) 7-ethyl-10-hydroxy-camptothecinKW-2170 (Kyowa Hakko) Antitumor dactinomycin (actinomycin D) bleomycinicacid antibiotics amonafide idarubicin doxorubicin (adriamycin) bleomycinA azonafide rubidazone deoxyrubicin bleomycin B anthrapyrazoleplicamycinp valrubicin mitomycin C oxantrazole porfiromycin daunorubicin(daunomycin) MEN-10755 (Menarini) losoxantronecyanomorpholinodoxorubicin epirubicin GPX-100 (Gem Pharmaceuticals)bleomycin sulfate (blenoxane) mitoxantrone (novantrone) therarubicinAntimitotic paclitaxel RPR 109881A (Aventis) agents SB 408075(GlaxoSmithKline) ZD 6126 (AstraZeneca) docetaxel TXD 258 (Aventis)E7010 (Abbott) PEG-paclitaxel (Enzon) Colchicines epothilone B(Novartis) PG-TXL (Cell Therapeutics) AZ10992 (Asahi) vinblastine T900607 (Tularik) IDN 5109 (Bayer) IDN-5109 (Indena) Vincristine T 138067(Tularik) A 105972 (Abbott) AVLB (Prescient NeuroPharma) Vinorelbinecryptophycin 52 (Eli Lilly) A 204197 (Abbott) azaepothilone B (BMS)Vindesine vinflunine (Fabre) LU 223651 (BASF) BNP-7787 (BioNumerik)dolastatin 10 (NCI) auristatin PE (Teikoku Hormone) D 24851 (ASTAMedica)CA-4 prodrug (OXiGENE) rhizoxin (Fujisawa) BMS 247550 (BMS) ER-86526(Eisai) dolastatin-10 (NIH) mivobulin (Warner-Lambert) BMS 184476(BMS)combretastatin A4 (BMS) CA-4 (OXiGENE) cemadotin (BASF) BMS 188797 (BMS)isohomohalichondrin-B (PharmaMar) taxoprexin (Protarga) AromataseAminoglutethimide anastrazole inhibitors Exemestane YM-511 (Yamanouchi)Letrozole formestane atamestane (BioMedicines) Thymidylate pemetrexed(Eli Lilly) ZD-9331 (BTG) synthase nolatrexed (Eximias) CoFactor ™(BioKeys) inhibitors DNA trabectedin (PharmaMar) albumin + 32P (IsotopeSolutions) antagonists mafosfamide (Baxter International) O6 benzylguanine (Paligent) glufosfamide (Baxter International) thymectacin(NewBiotics) apaziquone (Spectrum edotreotide (Novartis)Pharmaceuticals) Farnesyltransferase arglabin (NuOncology Labs) perillylalcohol (DOR BioPharma) are inhibitors tipifarnib (Johnson & Johnson)BAY-43-9006 (Bayer) lonafarnib (Schering-Plough) Pump inhibitors CBT-1(CBA Pharma) tariquidar (Xenova) zosuquidar trihydrochloride (Elibiricodar dicitrate (Vertex) Lilly) MS-209 (Schering AG) Histonetacedinaline (Pfizer) depsipeptide (Fujisawa) acetyltransferasepivaloyloxymethyl butyrate (Titan) MS-275 (Schering AG) inhibitors SAHA(Aton Pharma) Metalloproteinase Neovastat (Aeterna Laboratories)marimastat (British Biotech) inhibitors CMT-3 (CollaGenex) BMS-275291(Celltech) Ribonucleoside gallium maltolate (Titan) triapine (Vion)reductase tezacitabine (Aventis) didox (Molecules for Health) inhibitorsTNF alpha virulizin (Lorus Therapeutics) CDC-394 (Celgene) agonists/revimid (Celgene) antagonists Endothelin A atrasentan (Abbott) ZD-4054(AstraZeneca) receptor YM-598 (Yamanouchi) antagonist Retinoic acidfenretinide (Johnson & Johnson) LGD-1550 (Ligand) receptor agonistsalitretinoin (Ligand) Immuno- Interferon norelin (Biostar) modulatorsdexosome therapy (Anosys) IRX-2 (Immuno-Rx) oncophage (Antigenics)BLP-25 (Biomira) pentrix (Australian Cancer PEP-005 (Peplin Biotech)Technology) MGV (Progenics) GMK (Progenics) synchrovax vaccines (CTLImimmo) ISF-154 (Tragen) beta.-alethine (Dovetail) adenocarcinomavaccine (Biomira) melanoma vaccine (CTL Immuno) cancer vaccine(Intercell) CLL therapy (Vasogen) CTP-37 (A VI BioPharma) p21 RASvaccine (GemVax) Hormonal and estrogens bicalutamide antihormonalPrednisone testosterone propionate; agents conjugated estrogensfluoxymesterone methylprednisolone flutamide ethinyl estradiolmethyltestosterone prednisolone octreotide chlortrianisendiethylstilbestrol aminoglutethimide nilutamide idenestrol megestrolleuprolide mitotane tamoxifen hydroxyprogesterone caproate P-04(Novogen) goserelin Toremofine medroxyprogesterone 2-methoxyestradiol(EntreMed) leuporelin dexamethasone testosterone arzoxifene (Eli Lilly)Photodynamic talaporfin (Light Sciences) motexafin agentsPd-bacteriopheophorbide (Yeda) gadolinium (Pharmacyclics) Theralux(Theratechnologies) hypericin lutetium texaphyrin (Pharmacyclics)Tyrosine Kinase imatinib (Novartis) C225 (ImClone) Inhibitors kahalide F(PharmaMar) ZD4190 (AstraZeneca) leflunomide (Sugen/Pharmacia) rhu-Mab(Genentech) CEP-701 (Cephalon) ZD6474 (AstraZeneca) ZD1839 (AstraZeneca)MDX-H210 (Medarex) CEP-751 (Cephalon) vatalanib (Novartis) erlotinib(Oncogene Science) 2C4 (Genentech) MLN518 (Millenium) PKI166 (Novartis)canertinib (Pfizer) MDX-447 (Medarex) PKC412 (Novartis) GW2016(GlaxoSmithKline) squalamine (Genaera) ABX-EGF (Abgenix) phenoxodiol ( )EKB-509 (Wyeth) SU5416 (Pharmacia) IMC-1C11 (ImClone) trastuzumab(Genentech) EKB-569 (Wyeth) SU6668 (Pharmacia) Miscellaneous SR-27897(CCK A inhibitor, Sanofi- gemtuzumab (CD33 antibody, Wyeth Ayerst)Agents Synthelabo) CCI-779 (mTOR kinase inhibitor, Wyeth) BCX-1777 (PNPinhibitor, BioCryst) PG2 (hematopoiesis enhancer, Pharmagenesis)tocladesine (cyclic AMP agonist, Ribapharm) exisulind (PDE V inhibitor,Cell Pathways) ranpirnase (ribonuclease stimulant, Alfacell) Immunol ™(triclosan oral rinse, Endo) alvocidib (CDK inhibitor, Aventis) CP-461(PDE V inhibitor, Cell Pathways) galarubicin (RNA synthesis inhibitor,Dong-A) triacetyluridine (uridine prodrug, Wellstat) CV-247 (COX-2inhibitor, Ivy Medical) AG-2037 (GART inhibitor, Pfizer) tirapazamine(reducing agent, SRI International) SN-4071 (sarcoma agent, SignatureBioScience) P54 (COX-2 inhibitor, Phytopharm) WX-UK1 (plasminogenactivator inhibitor, N-acetylcysteine (reducing agent, Zambon) Wilex)CapCell ™ (CYP450 stimulant, Bavarian TransMID-107.TM. (immunotoxin, KSNordic) Biomedix) R-flurbiprofen (NF-kappaB inhibitor, Encore) PBI-1402(PMN stimulant, ProMetic GCS-100 (gal3 antagonist, GlycoGenesys)LifeSciences) 3CPA (NF-kappaB inhibitor, Active Biotech) PCK-3145(apoptosis promotor, Procyon) G17DT immunogen (gastrin inhibitor,Aphton) bortezomib (proteasome inhibitor, Millennium) seocalcitol(vitamin D receptor agonist, Leo) doranidazole (apoptosis promotor,Pola) efaproxiral (oxygenator, Allos Therapeutics) SRL-172 (T cellstimulant, SR Pharma) 131-I-TM-601 (DNA antagonist, CHS-828 (cytotoxicagent, Leo) TransMolecular) TLK-286 (glutathione S transferaseinhibitor, PI-88 (heparanase inhibitor, Progen) Telik) eflomithine (ODCinhibitor, ILEX Oncology) trans-retinoic acid (differentiator, NIH)tesmilifene (histamine antagonist, YM PT-100 (growth factor agonist,Point BioSciences) Therapeutics) minodronic acid (osteoclast inhibitor,MX6 (apoptosis promotor, MAXIA) Yamanouchi) midostaurin (PKC inhibitor,Novartis) histamine (histamine H2 receptor agonist, apomine (apoptosispromotor, ILEX Oncology) Maxim) bryostatin-1 (PKC stimulant, GPCBiotech) indisulam (p53 stimulant, Eisai) urocidin (apoptosis promotor,Bioniche) tiazofurin (IMPDH inhibitor, Ribapharm) CDA-II (apoptosispromotor, Everlife) aplidine (PPT inhibitor, PharmaMar) Ro-31-7453(apoptosis promotor, La Roche) cilengitide (integrin antagonist, MerckKGaA) SDX-101 (apoptosis promotor, Salmedix) rituximab (CD20 antibody,Genentech) brostallicin (apoptosis promotor, Pharmacia) SR-31747 (IL-1antagonist, Sanofi-Synthelabo) ceflatonin (apoptosis promotor,ChemGenex)Additional combinations may also include agents which reduce thetoxicity of the aforesaid agents, such as hepatic toxicity, neuronaltoxicity, nephprotoxicity and the like.Screening Assays

The compounds of the present invention may also be used in a method toscreen for other compounds that bind to an IAP BIR domain. Generallyspeaking, to use the compounds of the invention in a method ofidentifying compounds that bind to an IAP BIR domain, the IAP is boundto a support, and a compound of the invention is added to the assay.Alternatively, the compound of the invention may be bound to the supportand the IAP is added.

There are a number of ways in which to determine the binding of acompound of the present invention to the BIR domain. In one way, thecompound of the invention, for example, may be fluorescently orradioactively labeled and binding determined directly. For example, thismay be done by attaching the IAP to a solid support, adding a detectablylabeled compound of the invention, washing off excess reagent, anddetermining whether the amount of the detectable label is that presenton the solid support. Numerous blocking and washing steps may be used,which are known to those skilled in the art.

In some cases, only one of the components is labeled. For example,specific residues in the BIR domain may be labeled. Alternatively, morethan one component may be labeled with different labels; for example,using 1125 for the BIR domain, and a fluorescent label for the probe.

The compounds of the invention may also be used as competitors to screenfor additional drug candidates or test compounds. As used herein, theterms “drug candidate” or “test compounds” are used interchangeably anddescribe any molecule, for example, protein, oligopeptide, small organicmolecule, polysaccharide, polynucleotide, and the like, to be tested forbioactivity. The compounds may be capable of directly or indirectlyaltering the IAP biological activity.

Drug candidates can include various chemical classes, although typicallythey are small organic molecules having a molecular weight of more than100 and less than about 2,500 Daltons. Candidate agents typicallyinclude functional groups necessary for structural interaction withproteins, for example, hydrogen bonding and lipophilic binding, andtypically include at least an amine, carbonyl, hydroxyl, ether, orcarboxyl group. The drug candidates often include cyclical carbon orheterocyclic structures and/or aromatic or polyaromatic structuressubstituted with one or more functional groups.

Drug candidates can be obtained from any number of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides. Alternatively, libraries of natural compounds in theform of bacterial, fungal, plant and animal extracts are available orreadily produced. Additionally, natural or synthetically producedlibraries and compounds are readily modified through conventionalchemical, physical and biochemical means.

Competitive screening assays may be done by combining an IAP BIR domainand a probe to form a probe:BIR domain complex in a first samplefollowed by adding a test compound from a second sample. The binding ofthe test is determined, and a change or difference in binding betweenthe two samples indicates the presence of a test compound capable ofbinding to the BIR domain and potentially modulating the IAP's activity.

In one case, the binding of the test compound is determined through theuse of competitive binding assays. In this embodiment, the probe islabeled with a fluorescent label. Under certain circumstances, there maybe competitive binding between the test compound and the probe. Testcompounds which display the probe, resulting in a change in fluorescenceas compared to control, are considered to bind to the BIR region.

In one case, the test compound may be labeled. Either the test compound,or a compound of the present invention, or both, is added first to theIAP BIR domain for a time sufficient to allow binding to form a complex.

Formation of the probe:BIR domain complex typically require Incubationsof between 4° C. and 40° C. for between 10 minutes to about 1 hour toallow for high-throughput screening. Any excess of reagents aregenerally removed or washed away. The test compound is then added, andthe presence or absence of the labeled component is followed, toindicate binding to the BIR domain.

In one case, the probe is added first, followed by the test compound.Displacement of the probe is an indication the test compound is bindingto the BIR domain and thus is capable of binding to, and potentiallymodulating, the activity of IAP. Either component can be labeled. Forexample, the presence of probe in the wash solution indicatesdisplacement by the test compound. Alternatively, if the test compoundis labeled, the presence of the probe on the support indicatesdisplacement.

In one case, the test compound may be added first, with incubation andwashing, followed by the probe. The absence of binding by the probe mayindicate the test compound is bound to the BIR domain with a higheraffinity. Thus, if the probe is detected on the support, coupled with alack of test compound binding, may indicate the test compound is capableof binding to the BIR domain.

Modulation is tested by screening for a test compound's ability tomodulate the activity of IAP and includes combining a test compound withan IAP BIR domain, as described above, and determining an alteration inthe biological activity of the IAP. Therefore in this case, the testcompound should both bind to the BIR domain (although this may not benecessary), and alter its biological activity as defined herein.

Positive controls and negative controls may be used in the assays. Allcontrol and test samples are performed multiple times to obtainstatistically significant results. Following incubation, all samples arewashed free of non-specifically bound material and the amount of boundprobe determined. For example, where a radiolabel is employed, thesamples may be counted in a scintillation counter to determine theamount of bound compound.

Typically, the signals that are detected in the assay may includefluorescence, resonance energy transfer, time resolved fluorescence,radioactivity, fluorescence polarization, plasma resonance, orchemiluminescence and the like, depending on the nature of the label.Detectable labels useful in performing screening assays in thisinvention include a fluorescent label such as Fluorescein, Oregon green,dansyl, rhodamine, tetramethyl rhodamine, texas red, Eu³⁺; achemiluminescent label such as luciferase; colorimetric labels;enzymatic markers; or radioisotopes such as tritium, I¹²⁵ and the like

Affinity tags, which may be useful in performing the screening assays ofthe present invention include be biotin, polyhistidine and the like.

Synthesis and Methodology

General methods for the synthesis of the compounds of the presentinvention are shown below and are disclosed merely for the purpose ofillustration and are not meant to be interpreted as limiting theprocesses to make the compounds by any other methods. Those skilled inthe art will readily appreciate that a number of methods are availablefor the preparation of the compounds of the present invention.

General Procedures

Several methods for preparing symmetrically or non-symmetrically bridgedcompounds represented by formula I and formula II may be envisioned.General methods are illustrated in Schemes 1 to 8 and Schemes 17 to 20,while specific examples are illustrated in schemes 9 to 16 and Schemes21 to 26.

Scheme 1 illustrates a general procedure for the preparation ofbis-alkynyl bridged compounds of formula 1. N-PG¹-2-hydroxyproline isdeprotonated with NaH and treated with propargyl bromide to provide theproline intermediate 1-i. Activation of the carboxylic acid of 1-i withpeptide coupling agents and treatment with a primary or secondary amine,and deprotection of PG¹ provides the amide intermediate 1-ii. Peptidecoupling of PG²(H)N(R³)CHCO₂H with 1-ii is effected by activation of thecarboxylic acid of PG²(H)N(R³)CHCO₂H with peptide coupling agents,followed by the addition of 1-ii to provide the fully protected amide,which may be further deprotected at PG² to provide amide 1-iii.Activation of the carboxylic acid of PG³(R¹)N(R²)CHCO₂H with peptidecoupling agents, followed by the addition of 1-iii to provide the amideintermedaite 1-iv. The bis-alkynyl bridging moiety is prepared byhomo-coupling of the alkyne moieties of 1-iv using an appropriatecatalyst system, and subsequent deprotection of PG³, to provide compound1-v.

As illustrated in Scheme 2, intermediate 2-i is prepared via a typicalamide coupling/deprotection scheme. As such, the carboxylic acid moietyof PG¹-cis-2-amino-Pro(PG²)-OH is activated with amino acid couplingreagents, treated with an amine to provide the corresponding amide,followed by the removal of PG¹, under appropriate reaction conditions,to provide intermediate 2-i. In a similar manner, PG³(H)N(R³)HCCO₂H iscoupled with 2-i, followed by deprotection of PG³, to provide 2-ii.PG⁴(R¹)N(R²)HCCO₂H is coupled to 2-ii to provide 2-iii. Deprotection ofPG² provides 2-iv.

Scheme 3 illustrates that amide bridged compound of Formula I, may beprepared by the treatment of 3-i with an appropriately activated diacidin the presence of a base to give 3-ii. Deprotection of PG⁴ providescompounds of general formula 3-iii. Activation of the diacid may includethe use of active esters, acid chlorides, acid bromides, succinamideesters, HOBt esters, and the use of other reagents used in the formationof amide bonds.

Scheme 4 illustrates alkyl bridged compounds, which may be preparedusing the methods described herein. Treatment of 3-i with 0.5 equiv ofan alkyl chain containing two leaving groups, such as1,5-dibromopentane, 1,10-dibromodecane, and the like, providesintermediate 4-i. Alternatively, reductive amination of 3-i with adialdehyde may yield intermediate 4-i. Deprotection of PG⁴ yieldscompounds of formula 4-ii.

Scheme 5 illustrates one method wherein two BIR binding units may bebridged via a bis-acetylenic bridging unit. Coupling of 5-i with 5-iiprovides a mixture of the symmetrically bridged intermediates 5-iii and5-v, and the asymmetrical intermediate 5-iv. Separation of 5-ii, 5-iii,and 5-v, may be afforded by methods such as chromatography orrecrystallization. Deprotection of intermediates 5-iii, 5-iv, and 5-v,either independently or as a combined mixture, provides compounds 5-vi,5-vii, and 5-viii.

An additional method for the preparation of asymmetrically bridgedcompounds is illustrated in Scheme 6. Coupling of 5-i with 6-i willprovide a mixture of intermediates 6-ii, 5-iii and 6-iii. Separation of6-ii, 5-iii, and 6-iii, may be afforded by methods such aschromatography or recrystallization. Deprotection of PG⁴ ofintermediates 6-iii provides compound 6-iv.

An alternative strategy involves bridging two BIR binding units via abis-amide bridging group such as illustrated in Schemes 7 and 8.

The mono-protected bridging group HO₂C-L-CO₂PG⁵ is activated withpeptide coupling agents and subsequently treated with intermediate 3-ito provide intermediate 7-ii. Deprotection of PG⁵ provides intermediate7-iii. Treatment of 7-iii with peptide coupling agents, followed by7-iv, provides 7-v. Deprotection of PG⁴ and PG⁴⁰⁰ provides compound7-vi.

A similar process may be applied to the preparation of asymmetricallybridged BIR binding units, which have been bridged between P2 and P3, asillustrated in scheme 8. Treatment of 8-i with a cyclic anhydride, suchas succinic or glutaric anhydride, provides intermediate 8-ii. Treatmentof 8-ii with amide coupling agents followed by 3-i provides intermediate8-iii. Deprotection of PG⁴ provides compound 8-iv.

Scheme 9 illustrates the synthesis of compound 1.Boc-cis-2-hydroxy-L-proline was treated with NaH in DMF, followed bypropargyl bromide to provide intermediate 9-1. Amide coupling with(R)-1,2,3,4-tetrahydro-1-naphthlamine, and Boc deprotection with TFAprovided intermediate 9-2. Amide coupling of Boc-tert-BuGly-OH with 9-2,followed by Boc deprotection with TFA provided intermediate 9-3. Amidecoupling of Boc-MeAla-OH with intermediate 9-3 provided intermediate9-4. Homo-coupling of the acetylene groups of 9-4 using a Cul/TMEDAcatalyst under O₂, yielded intermediate 9-5, which was deprotected using4N HCl in 1-4,dioxane, to provide compound 1.2HCl.

Intermediate 10-6 was used in the preparation of compound 2 and 3 (seeSchemes 10 to 12). Intermediate 10-5, was prepared using a similarprocedure as described for intermediate 9-4, using Fmoc-AMPC(2S, 4S)—OHin the first step. Removal of the Fmoc protection group using a basesuch as 20% morpholine, in a solvent such as THF, provided intermediate10-6.

Treatment of intermediate 10-6 with 0.5 equiv of sebacoyl chloride inTHF provided 11-1. Removal of the Boc protecting groups using 4N HCl in1,4-dioxane provided compound 2.2HCl (Scheme 11).

Similarly, treatment of intermediate 10-6 with 0.5 equiv ofterephthaloyl chloride in THF provided 12-1. Removal of the Bocprotecting groups using 1N HCl in 1,4-dioxane provided compound 3.2HCl(Scheme 12).

Scheme 13 illustrates the preparation of compounds 4 and 5 Intermediates9-4 and 13-1 were coupled using a CuCl and TMEDA catalysts system, inacetone, under an oxygen atmosphere, to provide a mixture ofintermediates 9-5, 13-2, and 13-3. Separation by silica gelchromatography provided the individual intermediates. Intermediates 13-2and 13-3 were separately deprotected by treatment with 4N HCl in1,4-dioxane, to provide compounds 4.2HCl and 5.2HCl, respectively.

Scheme 14 illustrates the preparation of compound 6. Intermediates 9-4and 14-1 were coupled using a CuCl and TMEDA catalyst system, inacetone, under an oxygen atmosphere. Intermediate 14-2 was isolated fromthe resulting mixture by silica gel chromatography. Intermedaites 14-2was deprotected by treatment with 4N HCl in 1,4-dioxane, to providecompound 6.2HCl.

Scheme 15 illustrates the preparation of P2-P3 bridged compounds 7 and8. Intermediate 15-1 was treated with glutaric anhydride to provideintermediate 15-2. Treatment of 15-2 with HBTU, HOBt, and DIPEA,followed by the addition of 10-6 in DMF, provided intermediate 15-3.Removal of the Cbz protection group using H₂ over Pd/C yieldedintermediate 15-4. Acylation of 15-4 with Boc-N-MeAla-OH providedintermediate 15-5, which was Boc deprotected using 4N HCl in1,4-dioxane, provided compound 7.2HCl.

Removal of the Fmoc protection group from compound 7.2HCl, usingpolystyrene supported N-methylpiperazine in DMF provided compound 8.Removal of the Boc protection group from intermediate 15-3, using 4N HClin 1,4-dioxane, provided compound 9.HCl (see Scheme 16).

Scheme 17 illustrates the syntheses of compounds bridged by sulfonamidelinkages. Treatment of 3-i with a disulfonyl chloride reagent providesintermediate 17-i. Deprotection of PG⁴ provides compound 17-ii.

Similarly, treatment of 3-i with a bis-isocyanate provides intermediate18-i, which after deprotection of PG⁴ yields compound 18-ii.

Schemes 19a and 19b describe an alternate route to compounds 3-iii,17-ii or 18-ii, where A=CO and Q=NR⁴R⁵. Protected amino-prolinederivative 19-1 is treated with LG-L-LG to provide intermediate 19-2which is then deprotected at PG¹ to yield intermediate 19-3.Intermediate 19-3 is converted intermediate 19-5 by an amino acidcoupling/deprotection sequences as described earlier. A third amino acidcoupling step converts intermediate 19-5 to intermediate 19-6.Deprotection of 19-6 at PG² yields the diacid intermediate 19-7.Treatment of 19-7 with amino acid coupling reagents, followed R⁴R⁵NH,yields intermediate 19-8, which upon deprotection of PG⁴ providedcompound 19-9.

This method can be applied to the synthesis of bridged bis-prolineamides, sulfonamids and ureas. For example, in the case where thebridging group includes an Ar moiety, X-L-X═—NHS(O)₂—Ar—S(O)₂NH—,NHC(O)—Ar—C(O)NH—, or —NHC(O)NH—Ar—NHC(O)NH—, —O—CH₂ArCH₂—O—.

Alternatively, the proline derivative 19-2 may be deprotected at PG² andconverted to the amide intermediate 20-3. Following a similar procedureas described above, 20-3 can be converted to compound 19-9.

The synthesis of compound 20 is illustrated in Scheme 21.N-Boc-cis-4-amino-L-proline methyl ester, 21-1, was treated withterephthaloyl chloride to provide intermediate 21-2, which was furtherdeprotected with TFA to yield intermediate 21-3. Intermediate 21-3 wascoupled to Boc-tert-BuGyl-OH, 21-4, using HBTU and HOBt, followed by TFAdeprotection, to provide intermediate 21-6. Intermediate 21-6 wascoupled to Boc-N-MeAla-OH, 21-7, using HBTU and HOBt to provideintermediate 21-8. Saponification of the methyl ester using LiOHprovided intermediate 21-9. Coupling of intermediate 21-9 withphenethylamine using HBTU and HOBt provided intermediate 21-10 which wasdeprotected using HCl in 1,4-dioxane to provide compound 20.2HCl.

This method was used for the preparation of a number of proline amidederivatives such as compounds 19 to 24 and 46 to 51. This is shown inScheme 22 wherein, intermediate 21-9 was coupled to 2-propylamide,followed by HCl deprotection to yield compound 22.2HCl, while couplingof intermediate 21-9 to 1,1-diphenylmethylamine followed by deprotectionwith HCl provided compound 24.2HCl.

The preparation of pyrrolidine derivatives are described in Scheme 23.The ester moiety of intermediate 21-8 was reduced to alcohol 23-1 whichwas subsequently oxidized to aldehyde 23-2. Reductive amination usingphenethylamine provided intermediate 23-4. Acylation of 23-4 with eitheracetyl chloride or benzoyl chloride provided 23-5 and 23-6,respectively. Deprotection with 1N HCl in 1,4-dioxane provided compounds25.2HCl and 27.2HCl.

Sulfonylation of 23-4 with methanesulfonyl chloride, followed bydeprotection with 1 N HCl in 1,4-dioxane provided compound 26.2HCl.

Intermediate 24-1 provided an useful template for the preparation ofether bridged compounds. Intermediate 24-1 was prepared fromN-Boc-cis-4-hydroxy-L-proline and α,α′-dibromo-p-xylene as describedbelow. Amide coupling and deprotection of the Boc protecting groupsusing TFA provided intermediate 24-3. Two sequential amino acid couplingand deprotection steps, as described above, provided compound 29.2HCl.

Intermediate 10-6 was bridged using 1,3-benzenedisulfonyl chloride toprovide intermediate 25-1. Deprotection with 4N HCl in 1,4-dioxaneprovided compound 33 as its bis-HCl salt.

Similarly, intermediate 10-6 was bridged using 1,4-phenylenediisocyanate to provide intermediate 26-1, which upon deprotection withTFA provided compound 44 as is bis-TFA salt.

The preparation of compound 35 is illustrated in Scheme 27. Intermediate10-1 was deprotected by the treatment with piperide in THF to provideintermediate 27-1. Coupling of 27-1 with Cbz-Gly-OH followed by Bocdeprotection provided intermediate 27-3w TFA. Coupling of 27-3 withBoc-t-BuGly-OH followed by Boc deprotection provided intermediate27-5.TFA. Coupling of 27-5.2TFA with Boc-NMeAla-OH provided intermediate27-6. Removal of the Cbz group using H₂ and Pd/C yielded 27-7 which wasbridged using terephthaloyl chloride to provide intermediate 27-8.Deprotection of intermediate 27-8 using 4N HCl in 1,4-dioxane providedcompounds 35.2HCl.

Various chiral amines were prepared from optically active(R)-2-hydroxy-1-phenylethylamine, 28-1. Intermediate 28-1 was Bocprotected to provide 28-2. The alcohol moiety of 28-2 was alkylated withvarious alkyl halides to provide intermediates 28-3, 28-4, and 28-5.Deprotection of intermediates 28-3, 28-4 and 28-5 with HCl yielded thechiral amines 28-6, 28-7, and 28-8.

Intermediates 28-6, 28-7, and 28-8 were coupled with intermediate 21-9in a manner similar to that illustrated for compounds 22 and 24 (seeScheme 22) to provide compounds 63, 64 and 65, respectively.

Other chiral amines may be obtained commercially or prepared via anumber of methods including the conversion or interconversion ofketones, alcohols or azides to amines, using chiral or achiralchemistries, and the chiral resolution of isomers via methods known inthe art, such as chromatography or crystallization.

For example, Scheme 29 illustrates the enantioselective synthesis ofoptically enriched, asymmetric, 1,1-diphenylmethanamines prepared by theaddition of aryl boronic acids to chiral sulfinimines, followed byhydrolysis of the sulfinamines with acid to provide the opticallyenriched amine intermediates characterized by intermediates 29-4 and29-5 (Bolshan, Y.; Batey, R. A., Org. Letters 2005, 7, 1481-1484).

Intermediates 29-4 and 29-5 were coupled with intermediate 21-9 in amanner similar to that illustrated for compounds 22 and 24 (see Scheme22) to provide compounds 66 and 67, respectively.

Several methods exist for the conversion of tetralones to chiral1,2,3,4-tetrahydronaphthyl-1-amines, for example, the method report byR. A. Stalker, et al., Tetrahedron 2002, 58, 4837, as summarized below:

These chiral amines can be incorporated into compounds of the instantinvention using similar methods as illustrated in Schemes 21 and 22.

The above Schemes are applicable to both symmetrical compounds andunsymmetrical compounds of the present invention. The substituents A¹,A, Q, Q¹, R¹, R¹⁰⁰, R², R²⁰⁰, R³, R³⁰⁰, and the like are as definedherein. LG is a leaving group such as, for example, Cl, Br, I, OTs, OSuor OMs.

EXAMPLES

The following abbreviations are used throughout:

-   Boc: t-butoxycarbonyl;-   CBz: benzyloxycarbonyl;-   DCM: dichloromethane, CH₂Cl₂;-   DIPEA: diisopropylethylamine;-   DMAP: 4-(dimethylamino)pyridine;-   DMF: N,N-dimethylformamide;-   DTT: dithiothreitol;-   EDC: 3-dimethylaminopropyl-3-ethylcarbodiimide hydrochloride;-   EDTA: ethylenediaminetetracetic acid;-   Fmoc: N-(9-fluorenylmethoxycarbonyl);-   HBTU: O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium    hexafluorophosphate;-   HCl: hydrochloric acid;-   HOAc: acetic acid;-   HOBt: 1-hydroxybenzotriazole;-   HPLC: high performance liquid chromatography;-   LCMS: liquid chromatography-mass spectrometer;-   MeOH: methanol;-   MgSO₄: magnesium sulfate;-   MS: mass spectrum;-   NaHCO₃: sodium hydrogen carbonate;-   Pd/C: palladium on carbon;-   TEA: triethylamine;-   THF: tetrahydrofuran; and-   TMEDA: N,N,N,N-tetramethylethylenediamine.

Synthetic Methods

Synthesis of compound 1

Step 1: Intermediate 9-1

NaH (440 mg, 11.0 mmol) was suspended in dry DMF (5 mL) under N₂ at 0°C. Boc-cis-2-hydroxyl-L-proline (1.00 g, 4.0 mmol) was dissolved in dryDMF (15 mL) and added dropwise to the suspension of NaH. The mixture wasleft to stir at 0° C. for 10 min. Propargyl bromide (560 μL, 5.0 mmol)was added dropwise to the solution. The mixture was stirred at 0° C. for1 h then quenched with H₂O. The contents were added to a separatoryfunnel along with EtOAc and 10% citric acid (until pH ˜2). The organiclayer was collected, dried and concentrated under reduced pressure.Flash chromatography (silica, hexanes/THF) yielded intermediate 9-1 asclear oil. MS (m/z) M+Na=292.

Step 2: Intermediate 9-2

Intermediate 9-1 (560 mg, 2.1 mmol), HOBt (444 mg, 2.9 mmol), EDC (563mg, 2.9 mmol) and DIPEA (1.46 mL, 8.4 mmol) were dissolved in drydichloromethane (10 mL) under N₂ and stirred for 10 min at roomtemperature. 1,2,3,4-(R)-Tetrahydronaphtyl-1-amine (368 μL, 2.5 mmol)was then added and the solution was left to stir for 24 h at RT. Thecontents were then added to a separatory funnel along with EtOAc andwashed with 10% citric acid (2×), saturated NaHCO₃ (2×) and brine. Theorganic layer was collected, dried and concentrated under reducedpressure. The product was treated with 5 ml of 50% CH₂Cl₂/TFA for 1 hrat room temperature. Volatiles were removed in vacuo and the residuetriturated with diethyl ether to provide intermediate 9-2.TFA. MS (m/z)M+1=299.

Step Three: Intermediate 9-3

Boc-t-Bu-Gly-OH (484 mg, 2.1 mmol), HOBt (375 mg, 2.4 mmol), HBTU (910mg, 2.4 mmol) and DIPEA (1.20 mL, 7 mmol) were dissolved in dry DMF (10mL) under N₂ and stirred for 10 min at room temperature. Intermediate9-2 (720 mg, 1.75 mmol) was then added and the solution was left to stirfor 24 h at room temperature. The contents were then added to aseparatory funnel along with EtOAc and washed with 10% citric acid (2×),saturated NaHCO₃ (2×) and brine. The organic layer was collected, driedand concentrated under reduced pressure. The resulting product wastreated with 10 mL of 50% CH₂Cl₂/TFA for 1 hr at room temperature.Volatiles were removed in vacuo and the residue triturated with diethylether to provide intermediate 9-3.TFA. MS (m/z) M+1=412.

Step 4: Intermediate 9-4

Boc-N-Me-Ala-OH (278 mg, 1.37 mmol), HOBt (227 mg, 1.49 mmol), EDC (293mg, 1.49 mmol) and DIPEA (796 μL, 4.6 mmol) were dissolved in drydichloromethane (10 mL) under N₂ and stirred for 10 min at RT.Intermediate 9-3-TFA (600 mg, 1.14 mmol) was then added and the solutionwas left to stir for 24 h at RT. EtOAc was added and the oraganic layerwas washed with 10% citric acid (2×), saturated NaHCO₃ (2×) and brine,dried over anhydrous MgSO₄, filtered and volatiles removed under reducedpressure. The product was purified by silica gel chromatography, elutingwith 10-100% hexanes/THF, to provide intermediate 9-4. MS (m/z)M+Na=619.

Step 5: Compound 1

Intermediate 9-4 (100 mg, 0.17 mmol), CuCl (25 mg, 0.25 mmol) andN,N,N,N-tetramethylethylenediamine (38 μL, 0.25 mmol) were suspended indry acetone (5 ml) and stirred at room temperature under an O₂atmosphere for 72 h. EtOAc was added and the oraganic layer was washedwith 10% citric acid (2×), saturated NaHCO₃ (2×) and brine, dried overanhydrous MgSO₄, filtered and volatiles removed under reduced pressure.The product was purified by silica gel chromatography, eluting with10-100% hexanes/THF, to provide intermediate 9-5. Treatment of 9-5 with4N HCl in 1,4-dioxane at 0° C., for 2 hrs, and removal of volatilesunder reduced pressure provided compound 1 as its bis-HCl salt. MS (m/z)M+1=991.7.

Synthesis of Intermediate 10-6

Step 1: Intermediate 10-1

Fmoc-AMPC(2S,4S)—OH (900 mg, 2.0 mmol), HBTU (1.14 g, 3.0 mmol), andHOBt (415 mg, 3.0 mmol) were dissolved in dry DMF (10 mL) and treatedwith DIPEA (1050 uL, 6.0 mmol). The mixture was stirred for 10 minutesbefore the addition of (R)-1,2,3,4-tetrahydronaphthyl-1-amine (330 uL,2.2 mmol). The reaction was stirred overnight before being diluted withethyl acetate (100 mL) and 10% citric acid (50 mL). The organic layerwas separated and washed with 10% citric acid (2×50 mL), saturatedsodium bicarbonate (3×25 mL) and brine (1×20 mL), before being driedover anhydrouns magnesium sulphate, filtered, and concentrated underreduced pressure to provide crude 10-1 a white solid. This material was95% pure by LCMS and was advanced to the next step without furtherpurification.

Step 2: Intermediate 10-2

Intermediate 10-1 was dissolved in methylene chloride (10 mL) andtreated with TFA (10 mL). The solution was stirred at room temperaturefor 2 hrs before the volatiles were removed under reduced pressure. Theresulting oil was stirred with diethyl ether (25 mL) to provide a lightbrown solid which was filtered and washed with diethyl ether (2×5 mL),to provide compound 10-2.TFA as a light brown solid (1.17 g).

Step 3: Intermediate 10-3

Boc-t-BuGly-OH (460 mg, 2.0 mmol), HBTU (760 mg, 2.0 mmol), and HOBt(270 mg, 2.0 mmol) and DIPEA (765 uL, 7.5 mmol) were dissolved in dryDMF (10 mL) and the reaction was stirred at room temperature for 10minutes before intermediate 10-2 (867 mg, 1.5 mmol) was added. Themixture was stirred overnight before being diluted with ethyl acetate(200 mL) and 10% citric acid (50 mL). The organic layer was separatedand washed with 10% citric acid (2×50 mL), saturated sodium bicarbonate(3×25 mL) and brine (1×20 mL), before being dried over anhydrousmagnesium sulphate, filtered, and concentrated under reduced pressure toprovide crude 10-3 a white solid (920 mg, >95% pure by LCMS) and wasadvanced to the next step without further purification.

Step 4: Intermediate 10-4

Intermediate 10-3 was dissolved in methylene chloride (10 mL) andtreated with TFA (10 mL). The solution was stirred at room temperaturefor 2 hrs before the volatiles were removed under reduced pressure. Theresulting oil was stirred with diethyl ether (20 mL) to provide a lightbrown solid which was filtered, wash with diethyl ether (2×5 mL), toprovide compound 10-4.TFA a white solid.

Step 5: Intermediate 10-5

Boc-MeAla-OH (308 mg, 1.52 mmol), HBTU (450 mg, 1.91 mmol), and HOBt(260 mg, 1.91 mmol) were dissolved in dry DMF (10 mL). DIPEA (886 uL,5.0 mmol) was added and the reaction was stirred at room temperature for10 minutes before intermediate 10-4 (900 mg, 1.27 mmol) was added. Themixture was stirred overnight before being diluted with ethyl acetate(200 mL) and 10% citric acid (50 mL). The organic layer was separatedand washed with 10% citric acid (2×50 mL), saturated sodium bicarbonate(3×25 mL) and brine (1×20 mL), before being dried over anhydrousmagnesium sulphate, filtered, and concentrated under reduced pressure toprovide crude 10-5 a white solid (0.87 mg, 95.5% pure by LCMS) and wasadvanced to the next step without further purification.

Step 6: Intermediate 10-6

Intermediate 10-5 was dissolved in 20% morpholine/THF (10 mL) and thesolution was stirred at room temperature for 16 hrs. Volatiles wereremoved under reduced pressure to provide compound 10-6 a white solid.Further purification by silica gel chromatography provide 10-6 which was95% pure LCMS (MS (m/z) M+1=617.4.

Synthesis of Compound 2

Step 1: Intermediate 11-1

Crude 10-6 (200 mg, 0.251 mmol) was dissolved in THF (5 mL) and cooledto in an ice bath. DIPEA (50 uL, 0.275 mmol) and sebacoyl chloride (26uL, 0.125 mmol) were added and the reaction was stirred for 4 hours atroom temperature before being diluted with ethyl acetate (20 mL) andsaturated sodium bicarbonate. The organic layer was separated, washedwith brine, dried over anhydrous magnesium sulphate, filtered and thesolvent removed under reduced pressure. The resulting crude solid waspurified by silica gel chromatography, eluting with a 10-100% hexane/THFgradient, to provide 11-1 as a white solid (55 mg).

Step 2: Compound 2

Intermediate 11-1 (50 mg) was treated with 4N HCl in 1,4-dioxane (3 mL)and stirred for 3 hrs. Volatiles were removed under reduced pressure andthe resulting solid was triturated with diethyl ether (3×5 mL). Theresulting solid was dried under reduced pressure to provide compound2.2HCl as an off white solid (30 mg). MS (m/z) (M+2)/2=541.4.

Compound 3

Step 1: Intermediate 12-1

Crude 10-6 (200 mg, 0.251 mmol) was dissolved in THF (5 mL) and cooledto 4° C. on an ice bath. DIPEA (50 uL, 0.275 mmol) was added.Terephthaloyl chloride (26 uL, 0.125 mmol) was added and the reactionwas stirred for 16 hours before being diluted with ethyl acetate (20 mL)and saturated sodium bicarbonate. The organic layer was separated,washed with brine, dried over anhydrous magnesium sulphate, filtered andthe solvent removed under reduced pressure. The resulting crude solidwas purified by silica gel chromatography, eluting with a 25-100%hexanes/THF gradient, to provide 12-1 as a white solid (90 mg).

Step 2: Compound 3

Intermediate 12-1 (90 mg) was treated with 4N HCl in 1,4-dioxane (3 mL)and stirred for 3 hrs. Volatiles were removed under reduced pressure andthe resulting solid was triturated with diethyl ether, filtered andwashed with diethyl ether (3×5 mL). The resulting solid was dried underreduced pressure to provide compound 3.2HCl as an off white solid (40mg). MS (m/z) (M+2)/2=523.4.

Compounds 4 and 5

Intermediate 9-4 (130 mg, 0.21 mmol) and intermediate 13-1 (130 mg, 0.22mmol), CuCl (60 mg, 0.6 mmol) and tetramethylethylenediamine (90 μL, 0.6mmol) were suspended in dry acetone (15 mL) and stirred at roomtemperature under an O₂ atmosphere for 120 h. EtOAc was added and theorganic layer was washed with 10% citric acid (2×), saturated NaHCO₃(2×) and brine, dried over anhydrous Mg₂SO₄, filtered, and concentratedunder reduced pressure. The product was purified by silica gelchromatography, eluting with 10-100% hexanes/THF, to provideintermediates 9-5, 13-2 and 13-3. Intermediates 13-2 and 13-3 wereindependently treated with 4N HCl in 1,4-dioxane. Volatiles were removedand the resulting solids triturated with diethyl ether, filtered andwashed with diethyl ether, to provide compounds 4 and 5, respectively,as their bis-HCl salts.

Compound 4.2HCl: MS (m/s) M+1=993.5.

Compound 5.2HCl: MS (m/z) M+1=995.6.

Compound 6

Intermediate 9-4 (250 mg, 0.400 mmol), intermediate 14-1 (560 mg, 0.900mmol), CuCl (267 mg, 2.7 mmol) and tetramethylethylenediamine (405 μL,2.7 mmol) were suspended in dry acetone (10 mL) and stirred at roomtemperature under an O₂ atmosphere for 72 h. Celite was added and themixture filtered trough a pad of celite. EtOAc was added to the filtrateand the resulting organic layer was washed with 10% citric acid (2×),saturated NaHCO₃ (2×) and brine, dried over anhydrous Mg₂SO₄, filtered,and concentrated under reduced pressure. The product was purified bysilica gel chromatography, eluting with 10-100% hexanes/THF, to provideintermediate 14-2. Intermediate 14-2 was treated with 4N HCl in1,4-dioxane. Volatiles were removed and the resulting solids trituratedwith diethyl ether, filtered and washed with diethyl ether, to providecompound 6 as its bis-HCl salts. MS (m/s) (M+2)/2=514.4.

Compound 7

Step 1:

Intermediate 15-1 (1.0 g, 1.2 mmol), glutaric anhydride (190 mg, 1.7mmol) and DIPEA (836 μL, 4.8 mmol) were dissolved in dichloromethane (20mL) under N₂ at 0° C. A catalytic amount of DMAP was added and thesolution was stirred for 30 min on ice and 24 hrs at room temperature.EtOAc was added to the filtrate and the resulting organic layer waswashed with 10% citric acid (2×), saturated NaHCO₃ (2×) and brine, driedover anhydrous Mg₂SO₄, filtered, and concentrated under reduced pressureto provide intermediate 15-2 as a white solid.

Step 2:

Intermediate 15-2 (420 mg, 0.5 mmol), HOBt (85 mg, 0.63 mmol), HBTU (222mg, 0.60 mmol) and DIPEA (313 μL, 1.8 mmol) were dissolved in dry DMF (5ml) under N₂ and stirred for 10 min at RT. Intermediate 10-6 (250 mg,0.45 mmol) was added and the solution was left to stir for 24 hrs atroom temperate. EtOAc was added and the resulting organic layer waswashed with 10% citric acid (2×), saturated NaHCO₃ (2×) and brine, driedover anhydrous Mg₂SO₄, filtered, and concentrated under reducedpressure. The product was purified by silica gel chromatography, elutingwith 10-100% hexanes/THF, to provide intermediate 15-3.

Step 3:

Intermediate 15-3 (240 mg, 0.17 mmol), 10 wt % Pd/C (50 mg, 50% H₂O),were suspended in MeOH (10 mL) and stirred for 24 h at room temperatureunder a H₂ atmosphere (1 atm). The contents were then filtered on celiteand washed with MeOH. The filtrate was concentrated under reducedpressure to provide the intermediate 15-4 as a white solid.

Step 4:

BocNMe-Ala-OH (57 mg, 0.28 mmol), HOBt (44 mg, 0.33 mmol), HBTU (116 mg,0.31 mmol) and DIPEA (90 μL, 0.52 mmol) were dissolved in dry DMF (5 mL)under N₂ and treated with intermediate 15-4 (160 mg, 0.13 mmol). Afterstirring for 24 h at room temperature EtOAc was added and the resultingorganic layer was washed with 10% citric acid (2×), saturated NaHCO₃(2×) and brine, dried over anhydrous Mg₂SO₄, filtered, and concentratedunder reduced pressure. The product was purified by silica gelchromatography, eluting with 10-100% hexanes/THF, to provideintermediate 15-5. Intermediate 15-5 was stirred with 4N HCl in1,4-dioxane for 1 hr at room temperature. Volatiles were removed underreduced pressure to provide compound 7 as its HCl salt. MS(M+2)/2=618.0.

Compound 8:

Compound 7.HCl (100 mg, 0.08 mmol) is dissolved in DMF (1 mL) and addedto a CH₂Cl₂ (5 mL) suspension of piperazinomethyl polystyrene resin(0.86 mmol/g, 356 mg, 0.3 mmol). After shaking at room temperature for48 hrs an additional 200 mg of resin was added. The mixture was shakenfor 20 days at room temperature before being, filtered, washing withMeOH. Volatiles were removed under reduced pressure to provide compound8 a white solid. MS (m/z) M+1=1012.

Compound 9:

Intermediate 15-3 (10 mg) was treated with 4N HCl in 1,4-dioxane for 1hr. Volatiles were removed under reduced pressure to provide compounds 9as its HCl salt. MS (m/s) (M+2)/2=642.

Compounds 10, 11, 12, 13, 14, 15, 16, 39, 40, 41, 42, 43, 52, 55, 56,57, and 58 and were prepared in a similar manner to that described forcompound 2, wherein the corresponding sulfonyl chloride or activateddiacid was substituted for sebacoyl chloride in Step 1. MScharacterization of these compounds is summarized in Table 1.

Compound 17 was prepared in a similar manner to that described forcompound 29 using 1,2,3,4-(R)-tetrahydronaphthyl-1-amine in place ofphenethylamide. MS (m/s) (M+2)/2=510.4.

Compound 18:

Intermediate 21-8 was stirred in 4N HCl in 1,4-dioxane for 2 hrs.Volatiles were removed in vacuo and the residue was triturated withdiethyl ether to provide compound 18.2HCl as a white solid. MS (m/z)M+1=815.4.

Compounds 19, 21, 22, 23, 24, 31, 32, 46, 47, 48, 49, 50, 51, 54, 59,60, 61, 63, 64, 65, and 67 were prepared in a similar manner to thatdescribed for compound 20, wherein phenethylamine was substituted by thecorresponding amine in Step 6. MS characterization of these compounds issummarized in Table 1.

Compound 20

Step 1: Intermediate 21-2

To a solution of N-Boc-cis-4-amino-L-proline methyl ester, 21-1, (10.0g, 35.61 mmol) in CH₂Cl₂ cooled to 0° C. were sequentially added TEA(14.88 mL, 106.80 mmol), DMAP (10 mg) and terephthaloyl chloride (3.61g, 17.80 mmol) and the reaction was stirred overnight at roomtemperature. Water and ethyl acetate were added, the organic layer wasseparated, washed with 10% citric acid, aqueous NaHCO₃ and brine, driedover anhydrous MgSO₄, filtered and concentrated in vacuo. Purificationby silica gel chromatography provided intermediate 21-2 as a whitesolid.

Step 2: Intermediate 21-3.2TFA

Intermediate 21-2 (4.80 g, 7.76 mmol) was dissolved in a mixture ofCH₂Cl₂ (40 mL) and TFA (40 mL) at 0° C. The solution was stirred for 4hrs at room temperature. Volatiles were removed under reduced pressureand the residue was triturated with diethyl ether to provideintermediate 21-3.2TFA as a white solid. MS (m/z) M+1=419.2

Step 3: Intermediate 21-5

To a solution of Boc-α-tBuGly-OH, 21-4, (3.95 g, 17.07 mmol) in DMFcooled to 0° C. were sequentially added DIPEA (13.5 mL, 77.6 mmol), HOBt(2.62 g, 19.4 mmol) and HBTU (7.36 g, 19.4 mmol). After stirring for 10minutes intermediate 21-3.2TFA (5.02 g, 7.76 mmol) was added and thereaction mixture was stirred overnight at room temperature. Water andethyl acetate were added, the organic layer was separated, washed with10% citric acid, aqueous NaHCO₃ and brine, dried over anhydrous MgSO₄,filtered and concentrated in vacuo. Purification by silica gelchromatography provided intermediate 21-5 as a white solid.

Step 4: Intermediate 21-6.2TFA

Intermediate 21-5 (6.55 g, 7.76 mmol) was dissolved in a mixture ofCH₂Cl₂ (40 mL) and TFA (40 mL) at 0° C. The solution was stirred for 3hrs at room temperature. Volatiles were removed under reduced pressureand the residue was triturated with diethyl ether to provideintermediate 21-6.2TFA as a white solid. MS (m/z) M+1=645.4

Step 4: Intermediate 21-8

To a solution of Boc-NMe-Ala-OH, 21-7, (3.15 g, 15.51 mmol) in DMFcooled to 0° C. were sequentially added DIPEA (12.3 mL, 70.5 mmol), HOBt(2.38 g, 17.63 mmol) and HBTU (6.69 g, 17.63 mmol). After stirring for10 minutes intermediate 21-62TFA (6.15 g, 7.05 mmol) was added and thereaction mixture was stirred overnight at room temperature. Water andethyl acetate were added, the organic layer was separated, washed with10% citric acid, aqueous NaHCO₃ and brine, dried over anhydrous MgSO₄,filtered and concentrated in vacuo. Purification by silica gelchromatography provided intermediate 21-8 as a white solid.

Step 5: Intermediate 21-9

To a solution of intermediate 21-8 (6.20 g, 6.11 mmol) in THF (80 mL)and MeOH (8 mL) cooled to 0° C. was added 1N aqueous LiOH (30.5 mL) andthe reaction was stirred overnight at room temperature. PH was adjustedto 6 with 10% citric acid and ethyl acetate was added, the organic layerwas separated, washed with brine, dried over anhydrous MgSO₄, filteredand concentrated in vacuo to provide intermediate 21-9 as a white solid.

Step 6: Intermediate 21-10

To a solution of intermediate 21-9 (150 mg, 0.15 mmol) in DMF cooled to0° C. were sequentially added DIPEA (265 uL, 1.52 mmol), HOBt (51 mg,0.38 mmol) and HBTU (144 mg, 0.38 mmol). After stirring for 10 minutesphenethylamine (42 uL, 0.33 mmol) was added and the reaction mixture wasstirred overnight at room temperature. Water and ethyl acetate wereadded, the organic layer was separated, washed with 10% citric acid,aqueous NaHCO₃ and brine, dried over anhydrous MgSO₄, filtered andconcentrated in vacuo. Purification by silica gel chromatographyprovided intermediate 21-10 as a white solid.

Step 7: Compound 20.2HCl

4N HCl in 1,4-dioxane (3.0 mL) was added to intermediate 21-10 (145 mg,0.12 mmol) and the solution was stirred for 1 hr at 0° C. Volatiles wereremoved under reduced pressure and the residue was triturated withdiethyl ether to provide compound 20.2HCl as a white solid. MS (m/z)(M+2)/2=497.6.

Compound 22.2HCl

Step 1: Intermediate 22-1

To a solution of intermediate 21-9 (75 mg, 0.08 mmol) in DMF cooled to0° C. were sequentially added DIPEA (135 uL, 0.76 mmol), HOBt (26 mg,0.19 mmol) and HBTU (72 mg, 0.19 mmol). After stirring for 10 minutesisopropylamine (14 uL, 0.17 mmol) was added and the reaction mixture wasstirred overnight at room temperature. Water and ethyl acetate wereadded, the organic layer was separated, washed with 10% citric acid,aqueous NaHCO₃ and brine, dried over anhydrous MgSO₄, filtered andconcentrated in vacuo. Purification by silica gel chromatographyprovided intermediate 22-1 as a white solid.

Step 2: Compound 22.2HCl

N HCl in 1,4 dioxane (2.0 mL) was added to intermediate 22-1 (58 mg,0.05 mmol) and the solution was stirred for 2 hrs at 0° C. Volatileswere removed under reduced pressure and the residue was triturated withdiethyl ether to provide compound 22.2HCl as a white solid. MS (m/z)(M+2)/2=435.4.

Compound 24.2HCl

Step 1: Intermediate 22-2

To a solution of intermediate 21-9 (600 mg, 0.61 mmol) in DMF cooled to0° C. were sequentially added DIPEA (1.0 mL, 6.08 mmol), HOBt (205 mg,1.52 mmol) and HBTU (576 mg, 1.52 mmol). After stirring for 10 minutesaminodiphenylmethane (230 uL, 1.34 mmol) was added and the reactionmixture was stirred overnight at room temperature. Water and ethylacetate were added, the organic layer was separated, washed with 10%citric acid, aqueous NaHCO₃ and brine, dried over anhydrous MgSO₄,filtered and concentrated in vacuo. Purification by silica gelchromatography provided intermediate 22-2 as a white solid.

Step 2: Compound 24.2HCl

N HCl in 1,4 dioxane (1.9 mL) was added to intermediate 22-2 (660 mg,0.50 mmol) and the solution was stirred for 1 hr at 0° C. Volatiles wereremoved under reduced pressure and the residue was triturated withdiethyl ether to provide compound 24.2HCl as a white solid. MS (m/z)(M+2)/2=435.4.

Compound 25

Step 1: Intermediate 23-1

To a solution of intermediate 21-8 (1.10 g, 1.08 mmol) in THF cooled to0° C. was added lithium borohydride (118 mg, 4.86 mmol) and the reactionmixture was stirred for 3 hrs at room temperature. Ethyl acetate and 10%citric acid were added. The organic layer was separated, washed withaqueous NaHCO₃ and brine, dried over anhydrous MgSO₄, filtered andconcentrated in vacuo. Purification by silica gel chromatographyprovided intermediate 23-1 as a white solid. MS (m/z) M+1=959.6

Step 2: Intermediate 23-2

To a solution of intermediate 23-1 (284 mg, 0.29 mmol) in CH₂Cl₂ wasadded Dess Martin periodinane (314 mg, 0.74 mmol) and the reaction wasstirred for 5 hrs at room temperature. Aqueous NaHCO₃ was added, theorganic layer was separated, washed with brine, dried over anhydrousMgSO₄, filtered and concentrated in vacuo. Purification by silica gelchromatography provided intermediate 23-2 as a white solid.

Step 3: Intermediate 23-4

To a solution of intermediate 23-2 (282 mg, 0.29 mmol) in CH₂Cl₂ wasadded phenethylamine (82 uL, 0.65 mmol). After stirring for 30 min atroom temperature sodium triacetoxyborohydride (280 mg, 1.32 mmol) wasadded and the reaction mixture was stirred for 2 hrs. Saturated aqueousNaHCO₃ was added, the organic layer was separated, washed with brine,dried over anhydrous MgSO₄, filtered and concentrated in vacuo.Purification by silica gel chromatography provided intermediate 23-4 asa white solid. MS (m/z) M+1=1165.8

Step 4: Intermediate 23-5

To a solution of intermediate 23-4 (100 mg, 0.08 mmol) in CH₂Cl₂ cooledto 0° C. were sequentially added triethylamine (60 uL, 0.43 mmol) andacetyl chloride (14 uL, 0.19 mmol). The reaction was stirred for 2 hrsat room temperature before water and ethyl acetate were added. Theorganic layer was separated, washed with 10% citric acid, aqueous NaHCO₃and brine, dried over anhydrous MgSO₄, filtered and concentrated invacuo. Purification by silica gel chromatography provided intermediate23-5 as a white solid.

Step 5: Compound 25.2HCl

N HCl in 1,4-dioxane (3 mL) was added to intermediate 23-5 (80 mg, 0.06mmol) and the solution was stirred for 1 hr at room temperature.Volatiles were removed under reduced pressure and the residue wastriturated with diethyl ether to provide compound 25.2HCl as a whitesolid. MS (m/z) (M+2)/2=525.6.

Compound 26.2HCl

Step 1: Intermediate 23-5

To a solution of intermediate 23-4 (100 mg, 0.08 mmol) in CH₂Cl₂ cooledto 0° C. were sequentially added TEA (60 uL, 0.43 mmol) andmethanesulfonyl chloride (15 uL, 0.19 mmol) and the reaction was stirredfor 2 hrs at room temperature. Water and ethyl acetate were added, theorganic layer was separated, washed with 10% citric acid, aqueous NaHCO₃and brine, dried over anhydrous MgSO₄, filtered and concentrated invacuo. Purification by silica gel chromatography provided intermediate23-5 as a white solid.

Step 2: Compound 26.2HCl

4N HCl in 1,4-dioxane (3 mL) was added to intermediate 23-5 (79 mg, 0.06mmol) and the solution was stirred for 1 hr at room temperature.Volatiles were removed under reduced pressure and the residue wastriturated with diethyl ether to provide compound 26.2HCl as a whitesolid. MS (m/z) (M+2)/2=561.6.

Compounds 27, 28 and 30 were prepared in a similar manner to thatdescribed for compound 26, wherein for compound 27 and 28 acetylchloride and benzoyl chloride, respectively, were used in place ofmethanesulfonyl chloride.

Compound 27—MS (m/z) (M+2)/2=587.6.

Compound 28—MS (m/z) (M+2)/2=543.6.

Compound 30—MS (m/z) (M+2)/2=579.6.

Compound 29

Step 1: Intermediate 24-1

To a 1.0 M solution of NaHMDS in THF (21.6 mL, 21.6 mmol) cooled to 0°C. was slowly added a solution of N-Boc-cis-4-hydroxy-L-proline (2.50 g,10.8 mmol) in DMF. After stirring for 20 minutes at 0° C.α,α′-dibromo-p-xylene (1.37 g, 5.0 mmol) was added and the reactionmixture was stirred overnight at room temperature. Water and ethylacetate were added, the organic layer was separated, dried over MgSO₄,filtered and concentrated in vacuo. Purification by silica gelchromatography provided intermediate 24-1 as a white solid.

Step 2: Intermediate 24-2

To a solution of intermediate 24-1 (200 mg, 0.35 mmol) in DMF cooled to0° C. were sequentially added DIPEA (365 uL, 2.1 mmol), HOBt (132 mg,0.98 mmol) and HBTU (345 mg, 0.91 mmol). After stirring for 10 minutesphenethylamine (107 uL, 0.85 mmol) was added and the reaction mixturewas stirred overnight at room temperature. Water and ethyl acetate wereadded, the organic layer was separated, washed with 10% citric acid,aqueous NaHCO₃ and brine, dried over anhydrous MgSO₄, filtered andconcentrated in vacuo. Purification by silica gel chromatographyprovided intermediate 24-2 as a white solid.

Step 3: Intermediate 24-3.2TFA

Intermediate 24-2 (260 mg, 0.35 mmol) was dissolved in a mixture ofCH₂Cl₂ (3 mL) and TFA (3 mL). The solution was stirred for 1 hr at roomtemperature. Volatiles were removed under reduced pressure and theresidue was triturated with diethyl ether to provide intermediate24-3.2TFA as a white solid. MS (m/z) M+1=571.4

Step 4: Intermediate 24-4

To a solution of Boc-α-^(t)BuGly-OH (256 mg, 1.10 mmol) in DMF cooled to0° C. were sequentially added DIPEA (361 uL, 2.10 mmol), HOBt (175 mg,1.3 mmol) and HBTU (455 mg, 1.2 mmol). After stirring for 10 minutesintermediate 24-32TFA (230 mg, 0.46 mmol) was added and the reactionmixture was stirred overnight at room temperature. Water and ethylacetate were added, the organic layer was separated, washed with 10%citric acid, aqueous NaHCO₃ and brine, dried over anhydrous MgSO₄,filtered and concentrated in vacuo. Purification by silica gelchromatography provided intermediate 24-4 as a white solid.

Step 5: Intermediate 24-5.2TFA

Intermediate 24-4 (458 mg, 0.46 mmol) was dissolved in a mixture ofCH₂Cl₂ (3 mL) and TFA (3 mL). The solution was stirred for 30 minutes atroom temperature. Volatiles were removed under reduced pressure and theresidue was triturated with diethyl ether to provide intermediate24-5.2TFA as a white solid. MS (m/z) M+1=797.6

Step 6: Intermediate 24-6

To a solution of Boc-NMe-Ala-OH (119 mg, 0.58 mmol) in DMF cooled to 0°C. were sequentially added DIPEA (209 uL, 1.2 mmol), HOBt (91 mg, 0.67mmol) and HBTU (236 mg, 0.62 mmol). After stirring for 10 minutesintermediate 24-52TFA (250 mg, 0.24 mmol) was added and the reactionmixture was stirred overnight at room temperature. Water and ethylacetate were added, the organic layer was separated, washed with 10%citric acid, aqueous NaHCO₃ and brine, dried over anhydrous MgSO₄,filtered and concentrated in vacuo. Purification by silica gelchromatography provided intermediate 24-6 as a white solid.

Step 7: Compound 29.2HCl

N HCl in 1,4-dioxane (1.0 mL) was added to intermediate 24-6 (280 mg,0.24 mmol) and the solution was stirred for 1 hr at 0° C. Volatiles wereremoved under reduced pressure and the residue was triturated withdiethyl ether to provide compound 29.2HCl as a white solid. MS (m/z)M+1=967.6.

Compound 33

Step 1: Intermediate 25-1

To a solution of intermediate 10-6 (258 mg, 0.50 mmol) in DMF weresequentially added DIPEA (217 uL, 1.25 mmol) and 1,3-benzenedisulfonylchloride (69 mg, 0.25 mmol) and the reaction was stirred overnight atroom temperature. Water and ethyl acetate were added, the organic layerwas separated, washed with 10% citric acid, aqueous NaHCO₃ and brine,dried over anhydrous MgSO₄, filtered and concentrated in vacuo.Purification by silica gel chromatography provided intermediate 25-1 asa white solid.

Step 2: Compound 33.2HCl

N HCl in 1,4-dioxane (2 mL) was added to intermediate 25-1 (143 mg, 0.11mmol) and the solution was stirred for 1 hr at 0° C. Volatiles wereremoved under reduced pressure and the residue was triturated withdiethyl ether to provide compound 33.2HCl as a white solid. MS (m/z)(M+2)/2=559.5.

Compound 34:

Compound 34 was prepared in a similar manner to compound 20, whereinN-Boc-cis-4-amino-L-proline methyl ester was substituted withN-Boc-trans-4-amino-L-proline methyl ester in step 1 and phenethylaminewas substituted with 1,2,3,4-(R)-tetrahydronaphthyl-1-amine in Step 6.MS (m/z) M+1=1045.8.

Compound 35.2HCl

Step 1: Intermediate 27-1

Intermediate 10-1 (3.90 g, 6.68 mmol) was dissolved in THF (100 mL) andtreated with piperidine (5.0 mL, 68.2 mmol). The solution was stirredfor 16 hrs at room temperature before volatiles were removed underreduced pressure to provide a semi-solid which was suspended in MeOH (5mL) and filtered. The filtrate was concentrated, suspended in MeOH (5mL), filtered and the filtrate concentrated to provide intermediate 27-1as a semi-solid (80% pure).

Step 2: Intermediate 27-2

To a solution of carbobenzyloxyglycine (699 mg, 3.34 mmol) in DMF cooledto 0° C. were sequentially added DIPEA (2.40 mL, 13.9 mmol), HOBt (488mg, 3.61 mmol) and HBTU (1.37 g, 3.61 mmol). After stirring for 10minutes intermediate 27-1 (1.00 g, 2.78 mmol) was added and the reactionmixture was stirred overnight at room temperature. Water and ethylacetate were added, the organic layer was separated, washed with 10%citric acid, aqueous NaHCO₃ and brine, dried over anhydrous MgSO₄,filtered and concentrated in vacuo. Purification by silica gelchromatography provided intermediate 27-2 as a white solid.

Step 3: Intermediate 27-3.TFA

Intermediate 27-2 (1.42 g, 2.58 mmol) was dissolved in a mixture ofCH₂Cl₂ (15 mL) and TFA (15 mL). The solution was stirred for 1 hr atroom temperature. Volatiles were removed under reduced pressure and theresidue was triturated with diethyl ether to provide intermediate27-3.TFA as a white solid. MS (m/z) M+1=451.4

Step 4: Intermediate 27-4

To a solution of Boc-α-tBuGly-OH (668 mg, 2.89 mmol) in DMF cooled to 0°C. were sequentially added DIPEA (2.1 mL, 12.0 mmol), HOBt (488 mg, 3.61mmol) and HBTU (1.37 g, 3.61 mmol). After stirring for 10 minutesintermediate 27-3.TFA (1.36 g, 2.41 mmol) was added and the reactionmixture was stirred overnight at room temperature. Water and ethylacetate were added, the organic layer was separated, washed with 10%citric acid, aqueous NaHCO₃ and brine, dried over anhydrous MgSO₄,filtered and concentrated in vacuo. Purification by silica gelchromatography provided intermediate 27-4 as a white solid.

Step 5: Intermediate 27-5.TFA

Intermediate 27-4 (1.38 g, 2.08 mmol) was dissolved in a mixture of DCM(10 mL) and TFA (10 mL). The solution was stirred for 4 hrs at roomtemperature. Volatiles were removed under reduced pressure and theresidue was triturated with diethyl ether to provide intermediate27-5.TFA as a white solid. MS (m/z) M+1=564.4

Step 6: Intermediate 27-6

To a solution of Boc-NMe-Ala-OH (902 mg, 4.44 mmol) in DMF cooled to 0°C. were sequentially added DIPEA (3.50 mL, 20.2 mmol), HOBt (682 mg,5.05 mmol) and HBTU (1.92 g, 5.05 mmol). After stirring for 10 minutesintermediate 27-5.TFA (1.14 g, 1.68 mmol) was added and the reactionmixture was stirred overnight at room temperature. Water and ethylacetate were added, the organic layer was separated, washed with 10%citric acid, aqueous NaHCO₃ and brine, dried over anhydrous MgSO₄,filtered and concentrated in vacuo. Purification by silica gelchromatography provided intermediate 27-6 as a white solid.

Step 7: Intermediate 27-7

To a solution of intermediate 27-6 (925 mg, 1.24 mmol) in anhydrous MeOH(25 mL) and stirred under N₂ was added 10% Pd/C (125 mg). The reactionmixture was purged with H₂ and stirred for 1 hr. The reaction was thenfiltered through celite and the filtrate was concentrated in vacuo.Purification by silica gel chromatography provided intermediate 27-7 asa white solid. MS (m/z) M+1=615.4

Step 8: Intermediate 27-8

To a solution of intermediate 27-7 (150 mg, 0.25 mmol) in DCM cooled to0° C. were sequentially added TEA (100 uL, 0.73 mmol) and terephthaloylchloride (25.0 mg, 0.12 mmol) and the reaction was stirred overnight atroom temperature. Water and ethyl acetate were added, the organic layerwas separated, washed with 10% citric acid, aqueous NaHCO₃ and brine,dried over anhydrous MgSO₄, filtered and concentrated in vacuo.Purification by silica gel chromatography provided intermediate 27-8 asa white solid.

Step 9: Compound 35.2HCl

4N HCl in 1,4-dioxane (2 mL) was added to intermediate 27-8 (166 mg,0.12 mmol) and the solution was stirred for 1 hr at room temperature.Volatiles were removed under reduced pressure and the residue wastriturated with diethyl ether to provide compound 35.2HCl as a whitesolid. MS (m/z) (M+2)/2=580.6.

Compounds 36, 37, and 38 were prepared from intermediate 27-7, in asimilar manner to that described for compound 35, using oxalylychloride, isophthaloyl dichloride and 4,4′-biphenyldicarbonyl chloride,respectively, in place of phthaloyl chloride in Step 8

Compound 36—MS (m/z) (M+2)/2=542.6.

Compound 37—MS (m/z) (M+2)/2=580.6.

Compound 38—MS (m/z) (M+2)/2=618.6.

Compound 44

Step 1: Intermediate 26-1

To a solution of intermediate 10-6 (150 mg, 0.27 mmol) in THF weresequentially added TEA (112 uL, 0.81 mmol) and 1,4-phenylenediisocyanate (43 mg, 0.27 mmol) and the reaction was stirred at roomtemperature for 4 hrs. Volatiles were removed under reduced pressure andthe residue purified by silica gel chromatography to provideintermediate 26-1 as a white solid.

Step 2: Compound 44.2TFA

Intermediate 26-1 (148 mg, 0.12 mmol) was dissolved in a mixture ofCH₂Cl₂ (1.5 mL) and TFA (0.4 mL). The solution was stirred for 1 hr atroom temperature. Volatiles were removed under reduced pressure and theresidue was triturated with diethyl ether to provide compound 44.2TFA asa white solid. MS (m/z) (M+2)/2=538.4.

Compound 62:

Intermediate 21-9 was treated with 4N HCl in 1,4-dioxane for 2 hrs.Volatiles were removed under reduced pressure and the residue wastriturated with diethyl ether to provide compounds 62.HCl as an offwhite solid. MS (m/z) M+1=787.6.

Intermediate 28-6

Step 1: Intermediate 28-2

(S)-(+)-2-Phenylglycinol, 28-1 (1.64 g, 12.0 mmol) was dissolved inCH₂Cl₂ (90 mL). Boc₂O (2.84 g, 13.0 mmol) and DMAP (34 mg, 0.02 mmol)were added and stirred for 1 hour at room temperature. The reactionmixture was diluted with diethyl ether (200 mL) and 1 N HCl (100 mL).The organic layer was washed with 1 M HCl (2×100 mL), dried overanhydrous MgSO₄, filtered and volatiles removed under reduced pressure.The resulting residue was purified by silica gel chromatography providedan oil to provide intermediate 28-2 as white solid (2.7 g, 95% yield).MS (m/z) M+1=238.2.

Step 2: Intermediate 28-6

Intermediate 28-2 (420 mg, 1.77 mmol) and iodomethane (330 μL, 5.29mmol) were dissolved in anhydrous DMF (25 mL). The mixture was cooled to0° C., and then NaH (60% dispersion in oil, 103 mg, 2.58 mmol) wasadded. After 2 hours, the reaction mixture was diluted with ethylacetate (200 mL) and 1 M HCl (100 mL). The organic layer was washed with1 M HCl (2×100 mL), dried over anhydrous MgSO₄, filtered and volatilesremoved under reduced pressure. The residue was purified by silica gelchromatography to provide intermediate 28-3 as an oil. Intermedaite 28-3was then chilled to 0° C. and treated with 4M HCl in 1,4-dioxane (5 mL).After stirring for 90 minutes volatiles were removed under reducedpressure and the resulting solid was washed with diethyl ether, toprovide intermediate 28-6-HCl as white solid (237 mg, 69% yield). MS(m/z) M+1=152.2.

A similar procedure was used for the preparation of intermediates 28-7and 28-8, wherein methyl iodide was replaced with benzyl bromide forintermediate 28-7 and iodoacetamide for intermediate 28-8.

Step 1: Intermediate 29-1

To a solution of benzaldehyde (840 μL, 8.25 mmol) in CH₂Cl₂ (150 mL) wasadded (S)-2-methylpropane-2-sulfinamide (1.0 g, 8.25 mmol) and titaniumethoxide (3.5 ml, 16.50 mmol). The reaction mixture was refluxed for 5hours and cooled to room temperature. Water was added and the mixturewas vigorously stirred, then filtered through celite. The aqueous layerwas extracted with CH₂Cl₂ (3×) and the combined organic extracts werewashed with brine, dried over anhydrous MgSO₄, filtered and concentratedin vacuo. Purification by silica gel chromatography providedintermediate 29-1 as a yellow oil.

Step 2: Intermediate 29-2

To a suspension of (S,E)-N-benzylidene-2-methylpropane-2-sulfinamide,29-11, (110 mg, 0.526 mmol), [Rh(cod)(CH₃CN)₂]BF₄ (20.1 mg, 0.053 mmol),para-tolylboronic acid (143 mg, 1.052 mmol) and Et₃N (147 μl, 1.052mmol) in dioxane (1.2 mL) was added H₂O (2.4 mL). The resulting brownsuspension was stirred for 2 days at room temperature. The aqueous layerwas extracted with EtOAc (3×) and the combined organic extracts werewashed with brine, dried over anhydrous MgSO₄, filtered and concentratedin vacuo. Purification by silica gel chromatography providedintermediate 29-2 as a white solid (d.e.=81%).

Step 3: Intermediate 29-4

To a solution of(S)-2-methyl-N—((R)-phenyl(p-tolyl)methyl)propane-2-sulfinamide (82 mg,0.27 mmol), 29-2, in MeOH (270 μL) was added 4N HCl in 1,4-dioxane 4(140 μL, 0.54 mmol). The solution was stirred for 1 hr at roomtemperature, then Et₂O was added and a white precipitate was formed. Theprecipitate was filtered and washed with Et₂O to provide intermediate29-4.HCl as white solid.

Step 4: Intermediate 29-5

To a solution of intermediate 21-9 (122 mg, 0.123 mmol) in DMF cooled to0° C. were sequentially added DIPEA (193 μL, 1.107 mmol), HOBt (42 mg,0.308 mmol) and HBTU (117 mg, 0.308 mmol). After stirring for 10 minutesintermediate 29-4.HCl (59 mg, 0.253 mmol) was added and the reactionmixture was stirred overnight at room temperature. Water and ethylacetate were added, the organic layer was separated, washed with 10%citric acid, aqueous NaHCO₃ and brine, dried over anhydrous MgSO₄,filtered and concentrated in vacuo. Purification by silica gelchromatography provided intermediate 66-1 as a pink solid.

Step 5: Compound 66.2HCl

4N HCl in 1,4-dioxane (1 ml) was added to intermediate 66-1 (135 mg,0.12 mmol). The solution was stirred for 1 hour at 0° C. Volatiles wereremoved under reduced pressure and the residue was triturated withdiethyl ether to provide compound 6.2HCl as a white solid. MS (m/z)(M+2)/2=573.6.

Representative compounds of the present invention were preparedaccording to the above procedures and are illustrated in Table 1:

TABLE 1 COM- POUND STRUCTURE MS 1

M + 1 = 991.7 [M + 2]/2 = 496.4 2

M + 1 = 1081.8 [M + 2]/2 = 541.4 3

[M + 2]/2 = 523.4 4

M + 1 = 993.5. 5

M + 1 = 995.6 (M + 2)/2 = 498.5 6

(M + 2)/2 = 514.4 7

(M + 2)/2 = 618.0 8

M + 1 = 1012 9

(M + 2)/2 = 642 10

[M + 2]/2 = 561.6 11

M + 1 = 997.6 [M + 2]/2 = 499.4 12

M + 1 = 1067.6 [M + 2]/2 = 534.5 13

[M + 2]/2 = 520.4 14

[M + 2]/2 = 506.4 15

M + 1 = 1073.6 [M + 2]/2 = 537.4 16

[M + 2]/2 = 569.4 17

M + 1 = 1019.6 [M + 2]/2 = 510.4 18

M + 1 = 815.4 [M + 2]/2 = 408.2 19

M + 1 = 1029.6 [M + 2]/2 = 515.4 20

[M + 2]/2 = 497.6 21

M + 1 = 1021.6 [M + 2]/2 = 511.6 22

M + 1 = 869.6 [M + 2]/2 = 435.4 23

M + 1 = 1045.8 [M + 2]/2 = 523.4 24

[M + 2]/2 = 559.4 25

[M + 2]/2 = 525.6 26

[M + 2]/2 = 561.4 27

[M + 2]/2 = 587.6 28

[M + 2]/2 = 543.6 29

M + 1 = 967.6 [M + 2]/2 = 484.5 30

M + 1 = 1157.6 [M + 2]/2 = 579.6 31

[M + 2]/2 = 573.6 32

[M + 2]/2 = 533.6 33

M + 1 = 1117.6 [M + 2]/2 = 559.5 34

M + 1 = 1045.8 [M + 2]/2 = 523.4 35

[M + 2]/2 = 580.6 36

[M + 2]/2 = 542.6 37

[M + 2]/2 = 580.6 38

[M + 2]/2 = 618.6 39

[M + 2]/2 = 597.4 40

M + 1 = 1045.6 [M + 2]/2 = 523.4 41

M + 1 = 1046.6 [M + 2]/2 = 523.8 42

[M + 2]/2 = 548.4 43

M + 1 = 1046.6 [M + 2]/2 = 523.8 44

[M + 2]/2 = 538.4 45

M + 1 = 1018.8 [M + 2]/2 = 509.6 46

M + 1 = 965.6 [M + 2]/2 = 483.4 47

M + 1 = 1113.8 [M + 2]/2 = 557.6 48

M + 1 = 1117.8 [M + 2]/2 = 559.6 49

M + 1 = 993.8 [M + 2]/2 = 497.6 50

M + 1 = 993.8 [M + 2]/2 = 497.6 51

[M + 2]/2 = 619.9 52

M + 1 = 1051.8 [M + 2]/2 = 526.6 53

M + 1 = 1046.8 [M + 2]/2 = 523.4 54

M + 1 = 1025.8 [M + 2]/2 = 513.5 55

M + 1 = 1046.8 [M + 2]/2 = 524.2 56

[M + 2]/2 = 584.6 57

M + 1 = 1095.8 [M + 2]/2 = 548.6 58

M + 1 = 1167.8 [M + 2]/2 = 584.6 59

M + 1 = 1025.8 [M + 2]/2 = 513.6 60

M + 1 = 1117.8 [M + 2]/2 = 559.6 61

M + 1 = 1017.8 [M + 2]/2 = 509.6 62

M + 1 = 787.6 [M + 2]/2 = 394.4 63

[M + 1] = 1053.8 (M + 2)/2 = 527.8 64

[M + 1] = 1206.0 (M + 2)/2 = 603.6 65

[M + 1] = 1139.8 (M + 2)/2 = 570.6 66

(M + 2)/2 = 573.6 67

Representative compounds of the present invention which can be preparedby simple modification of the above procedures are illustrated in Tables2 to 6:

TABLE 2

R⁴ R⁵ H

H

H

H —CH₃ H

H

H

H

H

TABLE 3 Formula 1A M1—BG—M2

M1 M2

TABLE 4 Formula 1B M1—BG—M2

M1 M2

TABLE 5

R¹, R², R³, R¹⁰⁰, R²⁰⁰ and R³⁰⁰ are defined as hereinabove, —X—L—X′ ischosen from:

R⁴ and R⁴⁰⁰ are H; R⁵ and R⁵⁰⁰ are chosen from:

wherein the aryl moieties may be substituted by R¹⁰ and wherein, R¹⁰ andR^(10′) are independently defined as R¹⁰ hereinabove, and wherein thealkyl may be further substituted by R⁶ as defined hereinabove.

TABLE 6

R¹, R², R³, R¹⁰⁰, R²⁰⁰ and R³⁰⁰ are defined as hereinabove, —X—L—X′ ischosen from:

R⁴ and R⁴⁰⁰ are H; R⁵ and R⁵⁰⁰ are chosen from:

wherein the aryl moieties may be substituted by R¹⁰ and wherein, R¹⁰ andR^(10′) are independently defined as R¹⁰ hereinabove, and wherein thealkyl may be further substituted by R⁶ as defined hereinabove.

Assays

Molecular Constructs for Expression

GST-XIAP BIR3RING: XIAP coding sequence amino acids 246-497 cloned intoPGEX2T1 via BamH1 and AVA I. The plasmid was transformed into E. coliDH5α for use in protein expression and purification.

GST-HIAP2 (cIAP-1) BIR 3: HIAP2 coding sequence from amino acids 251-363cloned into PGex4T3 via BamH1 and XhoI. The plasmid was transformed intoE. coli DH5α for use in protein expression and purification.

GST-HIAP1(cIAP-2) BIR 3: HIAP1 coding sequence from amino acids 236-349,cloned into PGex4T3 via BamH1 and XhoI. The plasmid was transformed intoE. coli DH5α for use in protein expression and purification.

GST-linker BIR 2 BIR3Ring: XIAP coding sequence from amino acids 93-497cloned into PGex4T1 via BamH1 and XhoI. Amino acids 93-497 wereamplified from full length XIAP in pGex4t3, using the primers:TTAATAGGATCCATCAACGGCTTTTATC and GCTGCATGTGTGTCAGAGG, using standard PCRconditions. The PCR fragment was TA cloned into pCR-2.1 (invitrogen).Linker BIR 2 BIR 3Ring was subcloned into pGex4T1 by BamHI/XhoIdigestion. The plasmid was transformed into E. coli DH5α for use inprotein expression and purification.

Full-length human XIAP, AEG plasmid number 23. XIAP coding sequenceamino acids 1-497 cloned into GST fusion vector, PGEX4T1 via BamH1 andXho I restriction sites. The plasmid was transformed into E. coli DH5αfor use in protein purification.

GST-XIAP linker BIR 2: XIAP linker BIR 2 coding sequence from aminoacids 93-497 cloned into pGex4T3 via BamHI and XhoI. The plasmid wastransformed into E. coli DH5α for use in protein expression andpurification.

Expression and Purification of Recombinant Proteins

A. Expression of Recombinant Proteins

Glutathione S-transferase (GST) tagged proteins were expressed inEscherichia coli strains DH5-alpha. For expression full length XIAP,individual or combinations of XIAP-BIR domains, cIAP-1, cIAP-2 and Livintransformed bacteria were cultured overnight at 37° C. in Luria Broth(LB) medium supplemented with 50 ug/ml of ampicillin. The overnightculture was then diluted 25 fold into fresh LB ampicillin supplementedmedia and bacteria were grown up to A₆₀₀=0.6 then induced with 1 mMisopropyl-D-1-thiogalactopyranoside for 3 hours. Upon induction, cellswere centrifuged at 5000 RPM for 10 minutes and the media was removed.Each pellet obtained from a 1 liter culture received 10 ml of lysisbuffer (50 mM Tris-HCl, 200 mM NaCl, 1 mM DTT, 1 mM PMSF, 2 mg/ml oflysosyme, 100 μg/ml)), was incubated at 4° C. with gentle shaking. After20 minutes of incubation, the cell suspension was placed at −80° C.overnight or until needed.

B. Purification of Recombinant Proteins

For purification of recombinant proteins, the IPTG-induced cell lysatewas thawed vortexed and then disrupted by flash freezing in liquidnitrogen two times with vortexing after each thaw. The cells weredisrupted further by passing the extract four times through a Bio-NebCell disruptor device (Glas-col) set at 100 psi with Nitrogen gas. Theextract was clarified by centrifugation at 4° C. at 15000 RPM in a SS-34Beckman rotor for 30 minutes. The resulting supernatant was then mixedwith 2 ml of glutathione-Sepharose beads (Pharmacia) per 500 ml cellculture (per 1000 ml culture for full length XIAP) for 1 hour at 4 C.Afterwards, the beads were washed 3 times with 1× Tris-Buffered Saline(TBS) to remove unbound proteins. The retained proteins were eluted with2 washes of 2 ml of 50 mM TRIS pH 8.0 containing 10 mM reducedglutathione. The eluted proteins were pooled and precipitated with 604g/liter of ammonium sulfate and the resulting pellet re-suspended intoan appropriate buffer. As judged by SDS-PAGE the purified proteinswere >90% pure. The protein concentration of purified proteins wasdetermined from the Bradford method.

His-tag proteins were expressed in the E. Coli strain in E. coli AD494cells using a pet28ACPP32 construct. The soluble protein fraction wasprepared as described above. For protein purification, the supernatantwas purified by affinity chromatography using chelating-Sepharose(Pharmacia) charged with NiSO₄ according to the manufacturer'sinstructions. Purity of the eluted protein was >90% pure as determinedby SDS-PAGE. The protein concentration of purified proteins wasdetermined from the Bradford assay.

Synthesis of Fluorescent Probe P1

A fluorescent peptide probe,Fmoc-Ala-Val-Pro-Phe-Tyr(t-Bu)-Leu-Pro-Gly(t-Bu)-Gly-OH was preparedusing standard Fmoc chemistry on 2-chlorotrityl chloride resin (Int. J.Pept. Prot. Res. 38:555-561, 1991). Cleavage from the resin wasperformed using 20% acetic acid in dichloromehane (DCM), which left theside chain still blocked. The C-terminal protected carboxylic acid wascoupled to 4′-(aminomethy)fluorescein (Molecular Probes, A-1351; Eugene,Oreg.) using excess diisopropylcarbodiimide (DIC) in dimethylformamide(DMF) at room temperature and was purified by silica gel chromatography(10% methanol in DCM). The N-terminal Fmoc protecting group was removedusing piperidine (20%) in DMF, and purified by silica gel chromatography(20% methanol in DCM, 0.5% HOAc). Finally, the t-butyl side chainprotective groups were removed using 95% trifluoroacetic acid containing2.5% water and 2.5% triisopropyl silane, to provide probe P1 (>95% pure,HPLC).

Binding AssayFluorescence Polarization-Based Competition Assay

For all assays, the fluorescence and fluorescence-polarization wasevaluated using a Tecan Polarion instrument with the excitation filterset at 485 nm and the emission filter set at 535 nm. For each assay, theconcentration of the target protein was first established by titrationof the selected protein in order to produce a linear dose-responsesignal when incubated alone in the presence of the fluorescent probe P1or P2. Upon establishing these conditions, the compounds potency (IC₅₀)and selectivity, was assessed in the presence of a fix defined-amount oftarget protein and fluorescent probe and a 10 point serial dilution ofthe selected compounds. For each IC₅₀ curve, the assays were run asfollowed: 25 uL/well of diluted compound in 50 mM MES buffer pH 6.5 wereadded into a black 96 well plate then 25 ul/well of bovine serum albumin(BSA) at 0.5 mg/ml in 50 mM MES pH 6.5. Auto-fluorescence for eachcompound was first assessed by performing a reading of the compound/BSAsolution alone. Then 25 uL of the fluorescein probe diluted into 50 mMMES containing 0.05 mg/ml BSA were added and a reading to detectquenching of fluorescein signal done. Finally 25 uL/well of the targetor control protein (GST-BIRs) diluted at the appropriate concentrationin 50 mM MES containing 0.05 mg/ml BSA were added and the fluorescencepolarization evaluated.

Determination of IC₅₀ and Inhibitory Constants

For each assay the relative polarization-fluorescence units were plottedagainst the final concentrations of compound and the IC₅₀ calculatedusing the Grad pad prism software and/or Cambridge soft. The ki valuewere derived from the calculated IC₅₀ value as described above andaccording to the equation described in Nikolovska-Coleska, Z. (2004)Anal Biochem 332, 261-273.

Fluorescence Polarization Competition Assay

The k_(i) of various compounds in the BIR2-BIR3-ring FP assay, usingprobe P2, was determined as described above. For example, compound 3displayed a k_(i) of less than 100 nM.

Caspase-3 Full Length XIAP, Linker BIR2 or Linker-BIR2-BIR3RINGDerepression Assay

In order to determine the relative activity of the selected compoundagainst XIAP-Bir2, we setup an in vitro assay where caspase-3 wasinhibited by GST fusion proteins of XIAP linker-Bir2, XIAP LinkerBir2-Bir3-RING or full-length XIAP. Caspase 3 (0.125 ul) and 12.25-34.25nM (final concentration) of GST-XIAP fusion protein (GST-Bir2,GST-Bir2Bir3RING or full-length XIAP) were co-incubated with serialdilutions of compound (200 uM-5 pM). Caspase 3 activity was measured byoverlaying 25 uL of a 0.4 mM DEVD-AMC solution. Final reaction volumewas 100 uL. All dilutions were performed in caspase buffer (50 mM HepespH 7.4, 100 mM NaCl, 10% sucrose, 1 mM EDTA, 10 mM DTT, 0.1% CHAPS(Stennicke, H. R., and Salvesen, G. S. (1997). Biochemicalcharacteristics of caspase-3, -6, -7, and -8. J. Biol. Chem. 272,25719-25723).

The fluorescent AMC released from the caspase-3 hydrolysis of thesubstrate was measured in a TECAN spectrophotometer at 360 nm excitationand 444 nm emission, after 15 minutes of incubation at room temperature.IC₅₀ values were calculated on a one or two-site competition model usingGraphPad v4.0, using the fluorescence values after 15 minutes ofincubation plotted against the log 10 concentration of compound.

IC₅₀ values of preferred compounds were shown to correlate with EC₅₀values against SKOV3s and were typically less that 1 uM.

Cell-Free Assay

Caspase De-Repression Assay Using Cellular Extracts (Apoptosome)

100 ug of 293 cell S100 extract and 0.25 uM-2 uM of GST-XIAP fusionprotein (XIAP-Bir3RING, XIAP-Bir2Bir3RING, or full-length XIAP) wereco-incubated with serial dilutions of compound (40 uM-5 pM). Caspasespresent in the extracts were activated by adding 1 mM dATP, 0.1 mM ALLN,133 ug Cytochrome C (final concentrations), and incubating at 37° C. for25 minutes. All reactions and dilutions used S100 buffer (50 mM Pipes pH7.0, 50 mM KCl, 0.5 mM EGTA pH 8.0, 2 mM MgCl2 supplemented with 1/1000dilutions of 2 mg/ml Cytochalisin B, 2 mg/ml Chymotstatin, Leupeptin,Pepstatin, Antipain, 0.1M PMSF, 1M DTT). Final reaction volume was 30ul. Caspase-3 activity was measured by overlaying 30 ul of a 0.4 mMDEVD-AMC solution. Released AMC cleavage was measured in a TECANspectrophotometer at 360 nm excitation and 444 nm emission, on a kineticcycle of 1 hour with readings taken every 5 minutes. Caspase activitywas calculated as V_(o) of AMC fluorescence/sec. Caspase de-repressionby our compounds was compared to fully activated extract and activatedextract repressed by the presence of XIAP fusion protein.

IC₅₀ values of preferred compounds were shown to correlate with EC₅₀values against SKOV3s and were typically less that 1 uM.

Cell Culture and Cell Death Assays

A. Cell Culture

MDA-MD-231 (breast) and H460 (lung) cancer cells were cultured inRPMI1640 media supplemented with 10% FBS and 100 units/mL of Penicillinand Steptomycin.

B. Assays

Survival assays were performed on various cell lines includingMDA-MB-231, SKOV3, H460, PC3, HCT-116, and SW480 cells. Cells wereseeded in 96 well plates at a respective density of 5000 and 2000 cellsper well and incubated at 37° C. in presence of 5% CO₂ for 24 hours.Selected compounds were diluted into the media at various concentrationranging from 0.01 uM up to 100 uM. Diluted compounds were added onto theMDA-MB-231 cells. For the MDA-MB-231 SKOV3, H460, PC3, HCT-116, andSW480 cells, the compounds were added either alone or in presence of 1-3ng/ml of TRAIL. After 72 hours cellular viability was evaluated by MTSbased assays. A solution of[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt; MTS] was added onto cells for a period of 1 to 4 hours. Uponincubation the amount of converted MTS was evaluated using a Tecanspectrophotometer set at 570 nm.

MDA-MB-231, SKOV3, and PC3 cells were treated with selected compounds ofthe present invention and found to have EC₅₀s of 100 nM or less. Whenthe above cell lines were treated with the compounds of the presentinvention in the presence of TRAIL, they were shown to have EC₅₀s of 50nM or less.

Survival MTT Assay

One day prior the treatment with compound, 2000 to 4000 cells per wellwere plated in a tissue culture treated 96 well format dish with 100 ulof media and incubated at 37° C., 5% CO₂. On the day of compoundtreatment, compounds were diluted with cell culture media to a workingstock concentration of 2×. 100 uL of diluted compound were then added toeach well. The treated plate was incubated for 72 h at 37° C., 5% CO₂.Upon incubation, the cell viability was assessed as followed 20 uL ofMTT reagent at 5 mg/ml were added per well to cell plate. The plate wasincubated for 2 h at 37° C. in presence of 5% CO₂. The supernatant wasthen removed from the plate and 100 uL of isopropanol were added. Theabsorbance was measured in a TECAN spectrophotometer at 570 nm. Thepercentage of viability was expressed in percentage of the signalobtained with non treated cells.

As seen in Table 7, compounds represented in Table 1 hereinabovegenerally displayed EC₅₀ values against MDA-MB-231 and SKOV-3 cells of<1 μM. Select compounds had EC₅₀ of <50 nM.

TABLE 7 MDA-MB231 SKOV-3 Compound EC₅₀ (nM) EC₅₀ (nM) 1 A A 2 A A 3 A A5 C 10 A A 11 A 12 A 13 A 14 A 15 A 16 A 17 A 18 D 19 A 20 A 21 A 22 B23 A 24 A 25 A 26 B 27 B 28 A 29 B 30 C 31 B 32 B 33 A 34 B 35 B 36 C 37C 38 B 39 A 40 A 41 A 42 A 43 B 44 A 45 D 46 B 47 A 48 B 49 A 50 B 51 B52 A 53 D 54 D 55 A 56 A 57 A 58 B 59 B 60 A 61 A 64 A A - EC₅₀ lessthan 50 nM B - EC⁵⁰ less than 250 nM C - EC₅₀ more than 1000 nM D- EC₅₀more than 1000 nMApoptosis Assay: Measurement of Caspase-3 Activity from Cultured Cells.

One day, prior to the treatment, 10,000 cells per well were plated in awhite tissue culture treated 96 well plate with 100 uL of media. On theday of compound treatment, compounds were diluted with cell culturemedia to a working stock concentration of 2× and 100 ul of dilutedcompound were added to each well and the plate was incubated for 5 h at37° C. in presence of 5% CO₂. Upon incubation, the plate was washedtwice with 200 uL of cold TRIS Buffered Saline (TBS) buffer. Cells werelysed with 50 ul of Caspase assay buffer (20 mM Tris-HCl ph 7.4, 0.1%NP-40, 0.1% Chaps, 1 mM DTT, 0.1 mM EDTA, 0.1 mM PMSF, 2 mg/mlChymostatin, Leupeptin, Pepstatin, Antipapin) then incubated at 4° C.with shaking for 30 minutes. 45 ul of Caspase assay buffer and 5 uL ofAc-DEVD-AMC at 1 mg/ml were added to each well, the plate shaken andincubated for 16 h at 37° C. The amount of release AMC was measured in aTECAN spectrophotometer at with the excitation and emission filter setat 360 nm and 444 nm. The percentage of Caspase-3 activity was expressedin comparison of the signal obtained with the non-treated cells.

IC₅₀ values of preferred compounds were shown to correlate with EC₅₀values against SKOV3s and were typically less that 1 uM.

Cellular Biochemistry:

A. Detection of XIAP and PARP/Caspase-3/Caspase-9

Detection of cell expressed XIAP and PARP were done by western blotting.Cells were plated at 300 000 cells/well in a 60 mm wells (6 wells platedish). The next day the cells were treated with selected compound at theindicated concentration. 24 hours later cells the trypsinized cells,pelleted by centrifugation at 1800 rpm at 4° C. The resulting pellet wasrinsed twice with cold TBS. The final washed pellet of cells was thelysed with 250 uL Lysis buffer (NP-40, glycerol, 1% of a proteaseinhibitor cocktail (Sigma)), placed at 4° C. for 25 min with gentleshaking. The cells extract was centrifuged at 4° C. for 10 min at 10,000rpm. Both the supernatant and the pellet were kept for western blottinganalysis as described below. From the supernatant, the protein contentwas evaluated and about 50 ug of protein was fractionated onto a 10%SDS-PAGE. Pellets were washed with the lysis buffer and re-suspend into50 ul of Lamelli buffer 1×, boiled and fractionated on SDS-PAGE. Uponelectrophoresis each gel was electro-transferred onto a nitrocellulosemembrane at 0.6 A for 2 hours. Membrane non-specific sites were blockedfor 1 hours with 5% Skim milk in TBST (TBS containing 0.1% (v/v)Tween-20) at RT. For protein immuno-detection, membranes were incubatedovernight with primary antibodies raised against XIAP clone 48 obtainedfrom Becton-Dickison) or PARP: obtained from Cell signal or caspase-3 orcaspase-9 primary antibodies were incubated at 4° C. with shaking atdilutions as follows:

-   -   XIAP clone 80 (Becton-Dickinson) . . . 1/2500    -   PARP (Cell Signal) . . . 1/2500    -   Caspase 3 (Sigma) . . . 1/1500    -   Caspase 9 (Upstate) . . . 1/1000        Upon overnight incubation, the membranes received three washes        of 15 min in TBST then were incubated for 1 hour at room        temperature in the presence of a secondary antibody coupled with        HRP-enzyme (Chemicon) and diluted at 1/5 000. Upon incubation        each membrane were washed three times with TBST and the        immunoreactive bands were detected by addition of a luminescent        substrate (ECL kit Amersham) and capture of signal on a X-RAY        film for various time of exposure.

Certain exemplified compounds were shown to induce the cleavage of PARPnear concentrations which correlate with EC₅₀ values against SKOV3s andwere typically less that 1 uM.

Hollow Fiber Model

Hollow fiber in vivo model were used to demonstrate in vivo efficacy ofselected compounds against selected cell lines as single agent therapyor in combination with selected cytotoxic agents. At day 1, selectedcell lines were cultured and the fiber filled at a cell density of about40,000 cells/fiber. At the day of operation (day 4), three fibers areimplanted sub-cutaneous into 28-35 Nu/Nu CD-1 male mice. On day 5, micestart to receive daily injection via sub-cutaneous route of controlvehicle or vehicle containing the selected compound at the appropriateconcentration and/or injection of cytotoxic agent via intra-peritonealroute. Upon 3-7 days of consecutive drug treatments, the animals aresacrificed, each fiber is removed and the metabolic viability of theremaining cells determined by MTT assay. Efficacy of the compound isdefine as the difference between the vehicle-treated animal and theanimal treated with the compound alone or the compound given incombination of the cytotoxic agent.

MDA-MB-231 cells were implanted on day 1. Compound 3 was administeredfor 4 consecutive days via IV bolus (tail vein) injections at 1, 3, and10 mg/kg (2 mg/mL in 20% aqueous HPCD). Complete suppression of cellgrowth, as compared to 20% HPCD control, was observed for compound 3 atdrug concentrations of 3 mg/kg.

SKOV-3 Human Ovarian Cancer Cell Line Xenograph Study with Compound 3

Female CD-1 nude mice (approximately 20-25 g) were subcutaneouslyinjected 5×10⁶ SKOV-3 human ovarian tumor cells in 50% matrigelsubcutaneously in the right flank. On day 55, when tumors wereapproximately 100 mm³, treatment was initiated with compound 3 on a 5on/2 off treatment schedule for the duration of the experiment. Tumorsize was measured with digital calipers and calculated as V=(a×b²)/2,wherein, a is the longest dimension and b is the width.

Tumor regression was observed while dosing compound 3 at 1 mg/kg whiletumor stasis was observed while dosing compound 3 at 0.3 mg/kg (see FIG.1).

MDA-MB-231 Human Mammary Cancer Cell Line Xenograph Study with Compound3

Female CD-1 nude mice (approximately 20-25 g) were subcutaneouslyinjected 1×106 MDA-MB-231 human mammary tumor cells in the right flank.On day 71, when tumors were approximately 90 mm³, treatment wasinitiated with compound 3 on a 5 on/2 off treatment schedule for theduration of the experiment. Tumor size was measured with digitalcalipers and calculated as V=(a×b²)/2, wherein, a is the longestdimension and b is the width.

Tumor regression was observed while dosing compound 3 at 1 mg/kg (seeFIG. 2).

Pharmacokinetic Studies

Selected compounds were dissolved into normal saline and given atvarious doses using different route of administration, includingintravenous bolus, intravenous infusion, oral and subcutaneousinjection.

Compound of the present invention demonstrated acceptablepharmacokinetics via various routes of administration.

In Vitro Potency

Compounds of the instant invention were shown to kill SKOV3 (ovarian),MDA-MB-231 (breast), BT549 (breast), HL-60 (acute promyelocyticleukemia) and PANC-1 (pancreatic), cell lines in vitro, demonstratingEC₅₀s ranging of 0.1 nM to 1000 nM (see Table 7).

The SAR of these compounds was mapped using SKOV3s and severalinteresting trends emerged. Changing the stereochemistry at thepyrrolidine bridging site effected the potency of the compounds as seenin the EC₅₀s of the cis- and trans-proline derivatives 3 and 29 (EC₅₀=1nM, 88 nM, respectively. Amide bridging units provided active compounds,however, a significant range in potency against SKOV3 cells was observedby varying the components of the bridging units. Small, conformationallyconstrained bridging units including, but not limited to, 1,4-phenyldicarboxamides (terephthaloylamides), 1,3-phenyl dicarboxamides,2,6-naphthyl dicarboxamides, 1,4-cyclohexyl dicarboxamides, 3,5-pyridyldicarboxamides, or C₂-C₁₀ aliphatic dicarboxamides provide highly activecompounds. Bridging units containing bis-glycine amides such ascompounds 30 provide less active compounds (EC₅₀=188 nM).

Ether, urea and sulfonamide bridging units provide compounds which wereactive against SKOV3 cells, although the sulfonamide bridged compoundswere generally less active.

Significant shifts in potency against SKOV3 cells were observed byvarying the P4 substitution, wherein (R)-stereochemistry at the R⁴/R⁴⁰⁰amide provides compounds which are 5-10 times more potent than thecorresponding (S)-isomer. The introduction of hydrophilic moieties closeto the amide provide less active compounds such as compound 49.

Additionally, alkylation of the N-terminal alanine moiety providescompounds which are up to 100 fold more potent than the correspondingunsubstituted N-terminal alanine derivative.

A subet of cancer cell lines, however, were not innately sensitive tocompounds of the instant invention, with EC₅₀s greater than 1000 nM. Wehave demonstrated that IAP BIR binding compounds demonstrate synergistickilling of various cancer cell lines with death receptor agonists suchof TRAIL, agonist TRAIL receptor anti-bodies, TNF-α, and others. Weherein disclose that compound of formula I and II also demonstratesynergistic killing of various cancer cell lines with death receptoragonists such of TRAIL. When these cells were treated with compound andTRAIL, or antagonist TRAIL antibody, these cell lines were highlysensitive to compound with EC₅₀s generally less than 100 nM.

These cancer cell lines include HELA (cervical), HCT116 (colon), PC3(prostate), OVCAR-3 (ovarian), HEY (ovarian), and H460 (lung). In thepresence of TRAIL (1-3 ng/mL) and varying concentrations of compound 3the EC₅₀s for the above cell lines were less than 1000 nM.

In Vivo Potency

Compound 3 was tested in SKOV3 and MDA-MB-231 xenograph tumour models(see FIGS. 1 and 2). In both cases tumour regression was observed at 1mg/kg when given 5 days on and 2 days off. Tumour stasis was observed inSKOV3 xenograph at 0.1 mg/kg.

Discussion

The above results suggest that the IAP BIR binding compounds of theinstant invention are highly potent agents, both in vitro and in vivo,wherein a mechanistic link between IAP binding and IAP modulations canbe correlated to anti-cancer efficacy. We have demonstrated thatcompounds of the instant invention bind to the BIR domains of the IAPswith high affinity resulting in the release of active caspases 3 and 9.Further, these compounds result in the induction of apoptosis in cancercells while synergistically sensitizing cancer cell lines to deathreceptor agonists such as TRAIL. Moreover, when tumour bearing animalswere treated with compounds of the instant invention demonstrated tumourstasis and/or tumour regression at pharmaceutically relavent doses.

Compounds of the instant invention demonstrated acceptablepharmacokinetics via several routes of administration.

Other Embodiments

From the foregoing description, it will be apparent to one of ordinaryskill in the art that variations and modifications may be made to theinvention described herein to adapt it to various usages and conditions.Such embodiments are also within the scope of the present invention.

All publications mentioned in this specification are hereby incorporatedby reference.

1. A compound having a structure: Cpd STRUCTURE 2

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2. A pharmaceutical composition comprising a compound of claim 1 and apharmaceutically acceptable carrier.
 3. A compound having a structure:


4. A compound having a structure:


5. A compound having a structure:


6. A compound having a structure:


7. A compound having a structure:


8. A pharmaceutical composition comprising a compound of claim 3 and apharmaceutically acceptable carrier.
 9. A pharmaceutical compositioncomprising a compound of claim 4 and a pharmaceutically acceptablecarrier.
 10. A pharmaceutical composition comprising a compound of claim5 and a pharmaceutically acceptable carrier.
 11. A pharmaceuticalcomposition comprising a compound of claim 6 and a pharmaceuticallyacceptable carrier.
 12. A pharmaceutical composition comprising acompound of claim 7 and a pharmaceutically acceptable carrier